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Biomedical subjects

D Merlo

Publications and source records attributed to D Merlo.

At least 19 recordsLinked to original sources
PubMed2007

Reduced GABAB receptor subunit expression and paired-pulse depression in a genetic model of absence seizures.

Neocortical networks play a major role in the genesis of generalized spike-and-wave (SW) discharges associated with absence seizures in humans and in animal models, including genetically predisposed WAG/Rij rats. Here, we tested the hypothesis that alterations in GABA(B) receptors contribute to neocortical hyperexcitability in these animals. By using Real-Time PCR we found that mRNA levels for most GABA(B(1)) subunits are diminished in epileptic WAG/Rij neocortex as compared with age-matched non-epileptic controls (NEC), whereas GABA(B(2)) mRNA is unchanged. Next, we investigated the cellular distribution of GABA(B(1)) and GABA(B(2)) subunits by confocal microscopy and discovered that GABA(B(1)) subunits fail to localize in the distal dendrites of WAG/Rij neocortical pyramidal cells. Intracellular recordings from neocortical cells in an in vitro slice preparation demonstrated reduced paired-pulse depression of pharmacologically isolated excitatory and inhibitory responses in epileptic WAG/Rij rats as compared with NECs; moreover, paired-pulse depression in NEC slices was diminished by a GABA(B) receptor antagonist to a greater extent than in WAG/Rij rats further suggesting GABA(B) receptor dysfunction. In conclusion, our data identify changes in GABA(B) receptor subunit expression and distribution along with decreased paired-pulse depression in epileptic WAG/Rij rat neocortex. We propose that these alterations may contribute to neocortical hyperexcitability and thus to SW generation in absence epilepsy.

Animals↗
PubMed2003

Cerebellar granule cell Cre recombinase expression.

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.

Animals↗
PubMed2003

Involvement of cholinergic and gabaergic systems in the fragile X knockout mice.

Fragile X syndrome is an inherited cause of mental retardation. We used extra- and intracellular recordings in brain slices obtained from wild type and fragile X knockout mice to establish whether bath application of the cholinergic agent carbachol (5 microM) induces different responses in neurons of the subiculum, a limbic structure involved in learning and memory. We found that carbachol diminished excitatory post-synaptic responses induced by CA1 stratum radiatum stimulation in wild type mice, but caused an unexpected increase in knockout animals. Moreover, these responses augmented in knockout mice after carbachol washout, a phenomenon that resembled the muscarinic long-term potentiation seen in wild type mice during application of carbachol and GABA(A) receptor antagonists. We also used paired-pulse stimulation to determine whether the changes in synaptic excitability induced by carbachol were caused by pre- or post-synaptic mechanism. Under control conditions, this protocol induced facilitation in both wild type and knockout mice; in contrast, during carbachol application, this facilitatory effect was seen in wild type mice only. In conclusion, our data highlight for the first time differences in cholinergic and GABA-ergic mechanisms that may contribute to the phenotype of fragile X patients.

Animals↗
PubMed2001

Expression of GABA(A) receptor subunits in rat brainstem auditory pathways: cochlear nuclei, superior olivary complex and nucleus of the lateral lemniscus.

Inhibition by GABA is important for auditory processing, but any adaptations of the ionotropic type A receptors are unknown. Here we describe, using in situ hybridization, the subunit expression patterns of GABA(A) receptors in the rat cochlear nucleus, superior olivary complex, and dorsal and ventral nuclei of the lateral lemniscus. All neurons express the beta3 and gamma2L subunit messenger RNAs, but use different alpha subunits. In the dorsal cochlear nucleus, fusiform (pyramidal) and giant cells express alpha1, alpha3, beta3 and gamma2L. Dorsal cochlear nucleus interneurons, particularly vertical or tuberculoventral cells and cartwheel cells, express alpha3, beta3 and gamma2L. In the ventral cochlear nucleus, octopus cells express alpha1, beta3, gamma2L and delta. Spherical cells express alpha1, alpha3, alpha5, beta3 and gamma2L. In the superior olivary complex, the expression profile is alpha3, alpha5, beta3 and gamma2L. Both dorsal and ventral cochlear nucleus granule cells express alpha1, alpha6, beta3 and gamma2L; unlike their cerebellar granule cell counterparts, they do not express beta2, gamma2S or the delta subunit genes. The delta subunit's absence from cochlear nucleus granule cells may mean that tonic inhibition mediated by extrasynaptic GABA(A) receptors is less important for this cell type. In both the dorsal and ventral nuclei of the lateral lemniscus, alpha1, beta3 and gamma2L are the main subunit messenger RNAs; the ventral nucleus also expresses the delta subunit. We have mapped, using in situ hybridization, the subunit expression patterns of the GABA(A) receptor in the auditory brainstem nuclei. In contrast to many brain regions, the beta2 subunit gene and gamma2S splice forms are not highly expressed in auditory brainstem nuclei. GABA(A) receptors containing beta3 and gamma2L may be particularly well suited to auditory processing, possibly because of the unique phosphorylation profile of this subunit combination.

Animals↗
PubMed2001

GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1.

Controlling the number of functional gamma-aminobutyric acid A (GABA(A)) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABA(A) receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABA(A) receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABA(A) receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABA(A) receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Antagonists of P2 receptor prevent NGF-dependent neuritogenesis in PC12 cells.

The pheochromocytoma PC12 cell line that develops neuronal characteristics of sympathetic cells after treatment with nerve growth factor (NGF) represents a well-established cellular model system for studying NGF signalling. Interesting information on the different mechanistic pathways of NGF can be obtained by adopting the pharmacological approach of inhibiting P2 receptors, expressed in naive PC12 cells and recognised as important biological mediators of neurotransmitters and growth factors. We show here that Basilen Blue, an antagonist of P2 receptor, reversibly prevents NGF-dependent neurite outgrowth with an IC(50) in the 5-10 microM range. Suramin, oxidised-ATP and diisothiocyanatostilbene-disulfonic acid, differently from other purinoceptor ligands, are also effective in this regard. NGF-dependent regeneration and stability of neurites, selected NGF-dependent extracellular and intracellular protein phosphorylations, binding of [(3)H] ATP to PC12 cell membranes are also modulated by Basilen Blue. On the contrary, cell adhesion, cellular duplication, 5'-nucleotidase activity, NGF-induced tyrosine autophosphorylation of TrkA receptors are not affected. NGF furthermore directly modulates the extracellular release of ATP and especially the levels of P2X(2) receptor protein in PC12 cells. In addition, extracellular ATP improves the neuritogenic effect of sub-optimal concentrations of NGF. Our study identifies P2 receptor ligands, particularly Basilen Blue, as useful tools to dissect different NGF-evoked functions, suggesting a mechanistic role for P2 receptors in the signalling pathways of NGF.

5'-Nucleotidase↗
PubMed1998

Mouse cerebellar granule cell differentiation: electrical activity regulates the GABAA receptor alpha 6 subunit gene.

GABAA receptor alpha6 subunit gene expression marks cerebellar granule cell maturation. To study this process, we used the Deltaalpha6lacZ mouse line, which has a lacZ reporter inserted into the alpha6 gene. At early stages of postnatal cerebellar development, alpha6-lacZ expression is mosaic; expression starts at postnatal day 5 in lobules 9 and 10, and alpha6-lacZ is switched on inside-out, appearing first in the deepest postmigratory granule cells. We looked for factors regulating this expression in cell culture. Membrane depolarization correlates inversely with alpha6-lacZ expression: granule cells grown in 25 mM [K+]o for 11-15 d do not express the alpha6 gene, whereas cultures grown for the same period in 5 mM [K+]o do. This is influenced by a critical early period: culturing for >/=3 d in 25 mM [K+]o curtails the ability to induce the alpha6 gene on transfer to 5 mM [K+]o. If the cells start in 5 mM [K+]o, however, they still express the alpha6-lacZ gene in 25 mM [K+]o. In contrast to granule cells grown in 5 mM [K+]o, cells cultured in 25 mM [K+]o exhibit no action potentials, mEPSCs, or mIPSCs. In chronic 5 mM [K+]o, factors may therefore be released that induce alpha6. Blockade of ionotropic and metabotropic GABA and glutamate receptors or L-, N-, and P/Q-type Ca2+ channels did not prevent alpha6-lacZ expression, but inhibition of action potentials with tetrodotoxin blocked expression in a subpopulation of cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

Endometrial ablation with a vaporizing electrode. II. Clinical outcome of a pilot study.

BACKGROUND: As a vaporizing electrode has been successfully used to treat submucous myomas, we evaluated its safety and efficacy in performing endometrial ablation. METHODS: Forty consecutive women with established menorrhagia with (n=26) and without (n=14) submucous myomas were enrolled in a prospective, noncomparative, pilot study. Hysteroscopic endometrial vaporization was performed with pure cutting current set at 200 watts. RESULTS: All procedures were completed without complications. Median (interquartile range, IQR) fluid deficit was 90 (0-200) ml and median (IQR) operating time 10 (7-12.5) min. A significant correlation was observed between operating time and fluid absorption (Spearman's test by ranks, r=0.47; p=0.002). The degree of difficulty of the operation was classified as none on 28 (70%) occasions, mild on 11 (27.5%) and moderate on one (2.5%). After a mean+/-s.d. follow-up of 20.3+/-2.4 months, amenorrhea or spotting were reported by 23 (57.5%) subjects, hypomenorrhea by 10 (25%), normal flows by six (15%), and menorrhagia by one (2.5%). The median (IQR) menstrual score calculated according to a pictorial blood loss assessment chart dropped from 282.5 (199-383) to 0 (0-15) (p<0.0001). Six (15%) subjects were very satisfied with the effect of surgery, 30 (75%) satisfied, two (5%) uncertain, one (2.5%) dissatisfied and one (2.5%) very dissatisfied. CONCLUSIONS: The vaporizing electrode seems to combine the benefits of the cutting loop (speed, efficacy and possibility of removing myomas) and the roller-ball electrode (safety and limited fluid absorption) while avoiding their respective disadvantages, and may be considered an interesting alternative in the hysteroscopic treatment of menorrhagia.

Adult↗
PubMed1998

Transvaginal ultrasonography versus uterine needle biopsy in the diagnosis of diffuse adenomyosis.

To assess the reliability of transvaginal ultrasonography and uterine needle biopsy, used singly or in combination, in the diagnosis of diffuse adenomyosis, a prospective study with pathological confirmation of the diagnosis was performed. A total of 102 premenopausal women scheduled for hysterectomy because of menorrhagia and/or pelvic pain underwent preoperative transvaginal ultrasonography. After removal of the uterus, a single full-thickness myometrial biopsy specimen was taken from along the median line in the upper third of the posterior uterine wall, using a 14-gauge Trucut needle. Adenomyosis was diagnosed by the sonographer by the presence of indistinctly demarcated heterogeneous myometrial areas with distorted echotexture, and by the pathologist when the distance between the lower border of the endometrium and the affected myometrial area was more than one-half of a low power field. The prevalence of adenomyosis was 28% (29/102 patients). The sensitivity and specificity of transvaginal ultrasonography were 82.7 and 67.1% respectively, compared with 44.8 and 95.9% for uterine needle biopsy. The positive predictive values of the two tests were 50.0 and 81.2% respectively, and the negative predictive values 90.7 and 81.4%, likelihood ratios of a positive test 2.5 and 10.9, likelihood ratios of a negative test 0.3 and 0.6, and kappa indexes of agreement 0.42 and 0.47. Combining the tests did not improve the overall diagnostic performance. Both transvaginal ultrasonography and uterine needle biopsy demonstrated suboptimal test characteristics.

Adult↗
PubMed1997

Characterization of an ecto-phosphorylated protein of cultured cerebellar granule neurons.

Previous work identified the phosphorylation by extracellular ATP of an endogenous 45-kDa protein substrate and established the presence of ecto-protein kinase activity associated with cultured cerebellar granule neurons (Volonté et al.: J Neurochem 63:2028-2037, 1994). In this work, we characterize such ecto-phosphorylated 45-kDa protein substrate and its association with the cellular membrane. The total radioactive content of the 45-kDa protein is stable for the first 15 min after phosphorylation, and decreases about 70% in 30 min and 90% in approximately 2 hr. Rinsing the cells after the phosphorylating reaction causes a 50% removal of the incorporated radioactivity. Glycosidic residues are present on the 45-kDa ecto-protein, which is held in position on the cellular membrane through a specific glycosyl-phosphatidylinositol anchor. The extracellular incorporation of phosphate on the 45-kDa protein is not modulated by agents interfering with cytoskeleton stability, such as colchicine and taxol, or by gangliosides. The extracellular phosphorylation occurs mostly on serine residues, since the phosphate ester linkage is unstable at high pH and only antibodies raised against phosphoserine are capable of recognizing the 45-kDa ecto-protein.

Animals↗
PubMed1996

Binding and functions of extracellular ATP in cultured cerebellar granule neurons.

Selected purinoceptor modulators were previously shown to prevent glutamate-evoked cytotoxicity in cerebellar granule neurons. In the same cellular system, we now identify and characterize the presence of P2 receptors. The binding of [3H]ATP to membranes of cerebellar granule neurons grows monotonically as a function of neuronal differentiation, is saturable and reaches steady state within 6 min. Scatchard plot of the equilibrium saturation data is curvilinear with a Kd value of 28 nM and a Bmax value of 87 pmol/ mg of protein for the high affinity binding sites and a Kd value of 1.5 microM and Bmax value of 1.2 nmol/mg of protein, for the more numerous low affinity binding sites. We also show that extracellular ATP increases the release, but not the uptake, of [3H]D-aspartate and that it furthermore potentiates the release of [3H]D-aspartate evoked by glutamate and KCI. ATP itself is released by cerebellar granule cultures and such release grows monotonically as a function of neuronal differentiation. These data are consistent with the role that ATP is believed to play as a cotransmitter for the central nervous system.

Adenine Nucleotides↗
PubMed1996

Selected P2 purinoceptor modulators prevent glutamate-evoked cytotoxicity in cultured cerebellar granule neurons.

Primary cultures of granule neurons derived from cerebella of postnatal rats are endowed with Glu receptors. Glu receptor agonists exert a trophic influence on differentiating granule cells but, with maturation, the cells become vulnerable to excitatory amino acids. Here we show that the P2 purinoceptor antagonist basilen blue abolishes in rat cerebellar granule neurons the cytotoxic action of glutamate with an IC50 in the 10-20 microM range. Within the same concentrations, basilen blue inhibits binding of [3H] ATP to cerebellar granule cells, glutamate-evoked release (but not uptake) of [3H] D-aspartate and Ca2+ uptake. Furthermore, the extracellular phosphorylation of a major 45-kDa endogenous ecto-protein substrate of cerebellar granule neurons is inhibited with an IC50 of about 1 microM. Similar effects are elicited by 5-adenylylimidodiphosphate, a P2 purinoceptor agonist, when supplied to the neurons for 8 days previously to the addition of glutamate. Our data point to the use of P2 purinoceptor modulators as novel elements for understanding and controlling glutamate-mediated excitatory neurotoxicity and neurotransmission. We suggest a possible involvement of P2 purinoceptors in these actions.

Adenosine Triphosphate↗
PubMed1994

LiCl promotes survival of GABAergic neurons from cerebellum and cerebral cortex: LiCl induces survival of GABAergic neurons.

Previous studies showed that LiCl promotes short-term survival of PC12 cells after NGF or serum deprivation. In the present work, we investigate the survival effect of lithium on cerebellar granule primary cultures. While the total population of cerebellar neurons, mainly granule cells, showed only a short-term survival (about 20 h) in the presence of LiCl, the survival of 65-100% of the GABAergic interneurons originating from cerebellum and cerebral cortex at two different developmental stages was prolonged by 1-2 weeks. Optimal activity was elicited between 5 and 7 mM LiCl. The action of lithium required the presence of serum and persisted also after medium renewal. By direct visual inspection, LiCl promoted neuronal survival without apparently altering the morphological differentiation of the cells. Our studies thereby suggest a means to obtaining enriched populations of GABAergic neurons.

Animals↗
PubMed1994

Identification of an ectokinase activity in cerebellar granule primary neuronal cultures.

Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-32P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for approximately 20 min. Unlike what was seen with [gamma-32P]ATP, in the presence of [32P]orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80-90% in the presence of 1 microM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO4(3-). Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 microM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.

Adenosine Triphosphate↗
PubMed1993

Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.

Cerebellar granule cells grown in the presence of a serum complex differentiate but are resistant to the lethal action of excitatory amino acids. When these cells are grown also in the presence of insulin-like growth factor I (IGF-I) they become fully susceptible to the toxic, lethal action of glutamate. The glutamate-sensitizing action of IGF-I is dependent on concentration (half-maximal effect at 2-4 ng/ml) and time (half-maximal effect at 2-4 days in vitro) and is paralleled by the appearance of functionally active, glutamate-activated, Ca2+ channels and of voltage-gated Na+ and late K+ channels. IGF-I-induced glutamate sensitivity is rapidly reversible (t1/2 = 30-60 min) after removal of this somatomedin. The action of IGF-I is not mimicked by IGF-II, nerve growth factor, basic or acidic fibroblast growth factor, platelet-derived growth factor, or tumor necrosis factor alpha. We postulate that the constitutive phenotype of cerebellar granule cells is glutamate-resistant and becomes responsive to excitatory amino acids under the action of epigenetic cues among which IGF-I may be one of those operative in vivo.

Animals↗
PubMed1993

Cerebellar neurones: differentiation and modulation of sensitivity to excitotoxic treatment.

The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C18 column. SDS-PAGE analysis of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate.

Animals↗