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Biomedical subjects

F Impagnatiello

Publications and source records attributed to F Impagnatiello.

13 recordsLinked to original sources
PubMed1999

The phenotypic characteristics of heterozygous reeler mouse.

Histological and behavioral traits are associated with reelin (Reln) haplo-insufficiency in heterozygous reeler mouse (rl+/-). These phenotypic traits are an approximately 50% decrease of brain Reln mRNA and Reln protein, an accumulation of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons in subcortical white matter, an age-dependent decrease in prepulse inhibition of startle (PPI), and neophobic behavior on the elevated plus-maze. Possible analogies between these rl+/- phenotypic traits and signs of psychosis vulnerability are discussed.

Aging↗
PubMed1999

Glucocorticoids stimulate inflammatory 5-lipoxygenase gene expression and protein translocation in the brain.

In the brain, the expression of 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of inflammatory leukotrienes, increases during aging. Antiinflammatory drugs are currently being evaluated for the treatment of aging-associated neurodegenerative diseases such as Alzheimer's disease. Although generally considered antiinflammatory, glucocorticoids, whose production also increases during aging, are not particularly effective in this disease. In human monocytes, 5-LO mRNA content increases on exposure to the synthetic glucocorticoid dexamethasone, which prompted us to hypothesize that glucocorticoids might increase 5-LO expression in the brain as well. We treated rats for 10 days either with corticosterone (implanted subcutaneously) or with dexamethasone (injected daily); they were killed on day 10 after pellet implantation or 24 h after the 10th dexamethasone injection. We found increased levels of 5-LO mRNA and protein in hippocampus and cerebellum of glucocorticoid-treated rats; 5-LO-activating protein (FLAP) mRNA content was not affected. Using western immunobloting, we also observed the concurrent translocation of 5-LO protein from cytosol to membrane, an indication of its activation. Thus, glucocorticoid-mediated up-regulation of the neuronal 5-LO pathway may contribute to rendering an aging brain vulnerable to degeneration.

5-Lipoxygenase-Activating Proteins↗
PubMed1998

A decrease of reelin expression as a putative vulnerability factor in schizophrenia.

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.

Age of Onset↗
PubMed1998

Reelin is preferentially expressed in neurons synthesizing gamma-aminobutyric acid in cortex and hippocampus of adult rats.

During embryonic development of brain laminated structures, the protein Reelin, secreted into the extracellular matrix of the cortex and hippocampus by Cajal-Retzius (CR) cells located in the marginal zone, contributes to the regulation of migration and positioning of cortical and hippocampal neurons that do not synthesize Reelin. Soon after birth, the CR cells decrease, and they virtually disappear during the following 3 weeks. Despite their disappearance, we can quantify Reelin mRNA (approximately 200 amol/ g of total RNA) and visualize it by in situ hybridization, and we detect the translated product of this mRNA by immunocytochemistry preferentially in gamma-aminobutyric acid (GABA)ergic neurons of adult rat cortex and hippocampus. In adult rat cerebellum, Reelin is expressed in glutamatergic neurons (granule cells). The translated product of this mRNA is readily exported from the granule cell somata to the parallel fibers, where it has been detected by electron microscopy in axon terminals located presynaptically to Purkinje cell dendrites.

Animals↗
PubMed1998

Simultaneous detection of glutamic acid decarboxylase and reelin mRNA in adult rat neurons using in situ hybridization and immunofluorescence.

The combination of in situ hybridization and immunocytochemical technique is an important tool to detail the biochemical phenotype of individual neurons. In this work, we have developed a double fluorescence method to show the presence of reelin mRNA in GABAergic cells. This was achieved by demonstrating the colocalization of glutamic acid decarboxylase67, the synthesizing enzyme for GABA, with the mRNA for reelin, a novel factor involved in brain development and possibly the maintenance of the synaptic organization of layered structures in adult brain. The results demonstrated that reelin is expressed primarily in GABAergic cells in the adult rat cerebrum, but not in the cerebellum.

Animals↗
PubMed1998

Changes in AMPA receptor-spliced variant expression and shift in AMPA receptor spontaneous desensitization pharmacology during cerebellar granule cell maturation in vitro.

Using appropriate internal standards, quantitative reverse transcripase-polymerase chain reaction (RT-PCR), and cerebellar granule cell (CG) in primary cultures we have quantified the expression of mRNAs encoding for GluR1-4 DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits during neuronal maturation in vitro. GluR1 is the mRNA that increases during CG maturation; the expression changes of the other GluR mRNAs are minimal and the translation products of these mRNAs change with a similar pattern. During CG maturation, there is an 8- to 10-fold increase in the GluR1 FLOP mRNA and a twofold increase in the expression of FLOP mRNA for GluR4 and GluR4C. The GluR1 FLIP mRNA increases, but by a smaller extent. We found that the GluR2 mRNA is completely edited at its Q/R site during CG maturation. The increase on the expression of GluR1 FLIP and FLOP and of GluR4 FLOP mRNA variants during development is associated with a 10-fold increase in AMPA-mediated Na+ currents and in the increased amplification of this current by 7-chloro-3-methyl-3,4 dihydro-2H-1,2,4 benzothiadiazine S-S-dioxide (IDRA21) or by 6-chloro-3,4 dihydro-3-(2-norbornen-5-yl)-7-sulfamoyl-1,2,4-benzothiadiazine 1,1 dioxide (cyclothiazide [CT]).

Alternative Splicing↗
PubMed1997

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide: a partial modulator of AMPA receptor desensitization devoid of neurotoxicity.

In cerebellar granule neurons of neonatal rats micromolar concentrations of 7-chloro-3-methyl-3,4-dihydro-2H-1,2, 4-benzothiadiazine S,S-dioxide (IDRA-21) and cyclothiazide, two negative modulators of the spontaneous agonist-dependent rapid desensitization of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA)-gated ion channels, facilitate AMPA receptor function by increasing the content of free cytosolic Ca2+ as measured by single-cell fura-2 acetoxymethyl ester (Fura-2) Ca2+-dependent fluorescence and intracellular Na+ measured with the sodium-binding bezofuran isophthalate acetoxymethyl ester fluorescence indicator. IDRA-21 increases intracellular Na+ transient with a threshold (5 microM) that is approximately 10 times higher and has an intrinsic activity significantly lower than that of cyclothiazide. By virtue of its low intrinsic activity, IDRA-21 elicits a free cytosolic Ca2+ transient increase that is shorter lasting than that elicited by cyclothiazide even when the drug is left in contact with cultured granule cells for several minutes. Additionally, while dose dependently, 5-25 microM cyclothiazide in the presence of AMPA is highly neurotoxic, IDRA-21 (up to 100 microM) is devoid of neurotoxicity. The neurotoxicity elicited by cyclothiazide persists in the presence of dizocilpine (an antagonist of N-methyl-D-aspartate-selective glutamate receptors) but is blocked by 2,3-dihydroxy-6-nitrosulfamoylbenzo[f]quinoxaline (a competitive AMPA receptor antagonist) and the 1-(aminophenyl)-4-methyl-7, 8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466; a noncompetitive AMPA receptor antagonist). Since the doses of IDRA-21 that enhance cognitive processes in rats and monkeys are several orders of magnitude lower than those required to elicit marginal neurotoxicity in cultured neurons, it can be surmised that IDRA-21 is a potent cognition-enhancing drug virtually devoid of neurotoxic liability because it acts as a partial negative allosteric modulator of AMPA receptor desensitization.

Animals↗
PubMed1997

Tolerance to diazepam and changes in GABA(A) receptor subunit expression in rat neocortical areas.

Long-term treatment with diazepam, a full allosteric modulator of the GABA(A) receptor, results in tolerance to its anticonvulsant effects, whereas an equipotent treatment with the partial allosteric modulator imidazenil does not produce tolerance. Use of subunit-specific antibodies linked to gold particles allowed an immunocytochemical estimation of the expression density of the alpha1, alpha2, alpha3, alpha5, gamma(2L&S) and beta(2/3) subunits of the GABA(A) receptor in the frontoparietal motor and frontoparietal somatosensory cortices of rats that received long-term treatment with vehicle, diazepam (three times daily for 14 days, doses increasing from 17.6 to 70.4 micromol/kg), or imidazenil (three times daily for 14 days, doses increasing from 2.5 to 10.0 micromol/kg). In this study, tolerance to diazepam was associated with a selective decrease (37%) in the expression of the alpha1 subunit in layers III-IV of the frontoparietal motor cortex, and a concomitant increase in the expression of the alpha5 (150%), gamma(2L&S) and beta(2/3) subunits (48%); an increase in alpha5 subunits was measured in all cortical layers. In the frontoparietal somatosensory cortex, diazepam-tolerant rats had a 221% increase in the expression of alpha5 subunits in all cortical layers, as well as a 35% increase in the expression of alpha3 subunits restricted to layers V-VI. Western blot analysis substantiated that these diazepam-induced changes reflected the expression of full subunit molecules. Rats that received equipotent treatment with imidazenil did not become tolerant to its anticonvulsant properties, and did not show significant changes in the expression of any of the GABA(A) receptor subunits studied, with the exception of a small decrease in alpha2 subunits in cortical layers V-VI of the frontoparietal somatosensory cortex. The results of this study suggest that tolerance to benzodiazepines may be associated with select changes in subunit abundance, leading to the expression of different GABA(A) receptor subtypes in specific brain areas. These changes might be mediated by a unique homeostatic mechanism regulating the expression of GABA(A) receptor subtypes that maintain specific functional features of GABAergic function in cortical cell layers.

Animals↗
PubMed1996

Reversible modification of GABAA receptor subunit mRNA expression during tolerance to diazepam-induced cognition dysfunction.

Benzodiazepines (BZs) that are endowed with full positive allosteric modulatory (FAM) activity on GABAA receptors cause anterograde amnesia in both animals and humans. In rats subjected to a delayed object recognition test, diazepam, endowed with FAM activity, exerted an amnesic action, whereas BZs endowed with partial allosteric modulatory (PAM) activity on GABAA receptors, such as imidazenil, failed to induce amnesia, even if administered at doses five times higher than those equipotent to a standard anticonvulsant dose of diazepam (17.6 mumol/kg/os). After discontinuation of 14 days' treatment with vehicle, diazepam, or imidazenil (three times daily with increasing doses starting from 17.6 mumol/kg/os for diazepam and 2.5 mumol/kg/os for imidazenil), we compared the persistence of tolerance to the amnesic effect of diazepam with the persistence of the changes in the context of four (alpha 1, alpha 5, gamma 2L, gamma 2S) GABAA receptor subunit mRNAs in the fronto-parietal motor (FrPaM) cortex and the hippocampus. Rats receiving the long-term treatment with diazepam developed a tolerance to the amnesic effect of this drug and showed a decrease (30-50%) in the expression of mRNAs encoding for alpha 1 gamma 2L, gamma 2S GABAA receptor subunits, an increase, by approximately 30%, of the expression of mRNA of the alpha 5 subunit in the FrPaM cortex and a decrease, by approximately 25%, in the expression of mRNA, of the alpha 1 subunit in the hippocampus. These changes of subunit mRNA expression and the tolerance to the amnesic effect of diazepam returned to control values 72 hr after termination of the long-term treatment with diazepam. No tolerance to the amnesic effect of diazepam and no changes in GABAA receptor subunit mRNA expression were found in rats undergoing long-term treatment with imidazenil.

Animals↗
PubMed1996

Modifications of gamma-aminobutyric acidA receptor subunit expression in rat neocortex during tolerance to diazepam.

We evaluated whether tolerance to the antagonism of bicuculine-induced seizures by diazepam is associated with changes (i) in the content of mRNAs encoding for gamma-aminobutyric acidA (GABAA) receptor subunits, (ii) in the expression density of these subunits, and (iii) in the 1,4-benzodiazepine binding site characteristics in discrete neocortical structures. We found that in diazepam-tolerant rats, the content of the mRNA encoding for the alpha 1 subunit of the GABAA receptor decreased in the frontoparietal motor (FrPaM) cortex and in the hippocampus (42% and 20%, respectively) but not in the frontoparietal somatosensory (FrPaSS) cortex, striatum, olfactory bulb, and cerebellum. In the FrPaM cortex, gamma 2S and gamma 2L subunit mRNA contents were also decreased (48% and 30%, respectively), whereas that of alpha 5 was increased (30%). In the FrPaM and FrPaSS cortices as well as in cerebellum of diazepam-tolerant rats, the content of alpha 2, alpha 3, alpha 6, beta 2, and delta subunit mRNA was unchanged, as was the content of alpha 2, alpha 5, gamma 1, and gamma 2S subunit mRNA in the hippocampus. Furthermore, the reduction in alpha 1 subunit mRNA content in the FrPaM cortex and the anticonvulsant tolerance to diazepam returned to control values 72 hr after termination of the protracted diazepam treatment. Rats receiving a treatment with imidazenil in doses equipotent and with a schedule identical to that of diazepam failed to exhibit tolerance to the anticonvulsant action of this drug or cross-tolerance to diazepam. In these rats, the content of mRNA encoding for alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, gamma 1, gamma 2S, gamma 2L, and delta GABAA receptor subunits failed to change in the FrPaM and FrPaSS cortices, in the hippocampus, and in the other brain areas that were studied in diazepam-tolerant rats. Although the density and affinity of [3H]flumazenil and [3H]imidazenil binding failed to change in the FrPaM and FrPaSS cortices of diazepam-tolerant rats, the expression density of alpha 1 subunit immunogold labeling decreased by 37%, whereas that of alpha 5, gamma 2L/S, and beta 2/3 increased by 158%, 50%, and 47%, respectively, in the FrPaM cortex, and the density of the alpha 5 subunit selectively increased (209%) in the FrPaSS cortex. In contrast, the immunogold labeling density of the alpha 1, alpha 5, gamma 2L/S, and beta 2/3 subunits failed to change in either the FrPaM or FrPaSS cortex of rats receiving protracted imidazenil treatment.

Animals↗
PubMed1996

Semiquantitative immunocytochemical analysis of GABAA receptor subunit expression in the rat neostriatum.

A semiquantitative immunogold technique was used to investigate the levels of expression of specific GABAA receptor subunits in different regions (dorsolateral, dorsomedial and ventromedial regions) of the rat striatum. The results indicate that the subunits studied can be classified into three groups on the basis of their labelling density in the striatum: alpha 1 and alpha 3 (labelling density of less than 100 gold particles per 1000 microns2), gamma 2 and delta (between 100 and 200 particles per 1000 microns2), and alpha 2 and beta 2/3 (more than 300 particles per 1000 microns2). The alpha 1 and alpha 3 subunits are about 35% more abundant in the dorsal than in the ventral striatum, while the beta 2/3 and gamma 2 subunits are about 40% more abundant in the medial than in the lateral striatum. The alpha 2 and delta subunits did not show significant regional differences in abundance. The present data are consistent with the possibility that there are regional variations in the relative abundances of different GABAA receptor subtypes in the rat striatum.

Animals↗
PubMed1995

Inhibitory effect of ODN, a naturally occurring processing product of diazepam binding inhibitor, on secretagogues-induced insulin secretion.

Diazepam binding inhibitor (DBI1-86) is a peptide that is present in large amounts in the intestine and pancreas and which inhibits glucose-stimulated insulin release from both perfused pancreas and isolated islets in low nanomolar concentrations. Here, DBI33-50 (also known as ODN, octadecaneuropeptide), one of the naturally occurring processing products of DBI1-86, and certain synthetic modified derivatives, have been shown to inhibit glucose and glibenclamide-stimulated insulin secretion from isolated rat islets and glibenclamide-stimulated insulin secretion from hamster-insulinoma (HIT-T15) beta-cell line. DBI17-50 (TTN; triakontatetraneuropeptide), another prominent processing product of DBI, had no effect. The 50% inhibitory concentration (IC50) for the effect of ODN on insulin secretion induced by 8.3 of 16.7 mM glucose was approximately the same: 5 to 6 nM. Moreover, ODN inhibited insulin release induced by 0.01 or 1 microM glibenclamide with a similar IC50 (8 to 10 nM) in both isolated pancreatic islets and in HIT-T15 beta-cells. At concentration up to 1 microM, ODN had no effect on insulin secretion induced by PACAP (pituitary adenylate cyclase polypeptide), BAYK 8644 (methyl-(1,4-dihydro-2,6-dimethyl-3-nitro-4,2-trifluoromethylphenyl) pyridine-5-carboxylate), and only marginally it affected IBMX-(isobutylmethylxanthine) induced insulin secretion. This indicates that ODN does not act directly on ATP-regulated K+ channels, voltage dependent Ca2+ channels or cAMP production. In contrast, ODN inhibited insulin secretion induced by sodium nitroprussiate in a manner that is independent from the presence of extracellular Ca2+. These results suggest that ODN or ODN-like peptide fragments of DBI, may inhibit glucose or glibenclamide-induced insulin secretion via a signaling pathway that regulate the cytoplasmic free Ca2+ concentration.

1-Methyl-3-isobutylxanthine↗