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Stefano Vicini

Publications and source records attributed to Stefano Vicini.

At least 19 recordsLinked to original sources
PubMed2006

Deletion of the GABA(A) receptor alpha1 subunit increases tonic GABA(A) receptor current: a role for GABA uptake transporters.

The loss of more than half the number of GABA(A) receptors yet lack of pronounced phenotype in mice lacking the gene for the GABA(A) alpha1 subunit is somewhat paradoxical. We explored the role of tonic GABA(A) receptor-mediated current as a target of compensatory regulation in the alpha1 knock-out (-/-) mice. A 62% increase of tonic current was observed in the cerebellar granule cells (CGCs) of alpha1-/- compared with wild-type (+/+) mice along with a 67% increase of baseline current variance. Examination of whole-cell currents evoked by low concentrations of GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol suggested no upregulation of alpha6 and delta subunit-containing GABA(A) receptors in the alpha1-/-, confirming previous biochemical studies. Single-channel current openings were on average 32% shorter in the alpha1-/- neurons. Single-channel conductance and frequency of opening were not different between genotypes. Tonic current induced by application of the GABA transporter GAT-1 blocker NO711 (1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride) was significantly larger in the alpha1-/-, suggesting an increase of ambient GABA concentration. Experiments done with a known concentration of extracellular GABA complemented by a series of biochemical experiments revealed a reduction of GAT activity in alpha1-/- without an identifiable reduction of GAT-1 or GAT-3 protein. We report increased tonic GABA(A) receptor-mediated current in the alpha1-/- CGCs as a novel compensatory mechanism. Our data establish a role for GABA transporters as regulators of neuronal excitability in this and relevant models and examine other tonic conductance-regulating mechanisms responsible for the adaptive response of the cerebellar network to a deletion of a major synaptic GABA(A) receptor subunit.

Animals↗
PubMed2006

NMDA receptor subtypes at autaptic synapses of cerebellar granule neurons.

We studied the action potential-evoked autaptic N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-), and NR2C subunit knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between days in vitro 8 (DIV8) and DIV13. Compared with +/+ cells at DIV13, NR2A-/- cells had smaller and NR2C-/- cells had larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A-/- neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was roughly 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A-/- and NR2C-/- neurons at DIV12. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C-/- mice. This was mainly the result of an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg(2+) sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C-/- cells. Thus solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.

Animals↗
PubMed2006

Termination of epileptiform activity by cooling in rat hippocampal slice epilepsy models.

Cooling has been shown to terminate experimentally induced epileptiform activity in models of epilepsy without causing injury to the cooled brain, suggesting that cooling could represent an approach to seizure control in intractable focal epilepsies. Here we sought to determine the most effective way to apply cooling to abort spontaneous epileptiform discharges in in vitro brain slice models. We induced spontaneous epileptiform activity in rat brain slices by exposure to 4-aminopyridine (4-AP), 4-AP plus bicuculline, and Mg(2+)-free artificial CSF (aCSF) at 28-34 degrees C. Extracellular field recordings were made at hippocampal or neocortical sites. Slice temperature was reduced by perfusion with cold aCSF. Rapid cooling at rates of 2-5 degrees C/s was compared to cooling at slower rates of 0.1-1 degrees C/s. Cooling at both rates reversibly aborted epileptiform discharges in all three models and at all recording sites. With rapid cooling, small temperature drops were highly effective in terminating discharges, an effect that was sustained for as long as the reduced temperature level was maintained. In contrast, slow cooling required much larger temperature drops to inhibit discharges. With slow cooling, absolute temperature drops to 21-22 degrees C caused a 90% reduction in event frequency, but cooling to 14-15 degrees C was required to terminate discharges. We conclude that rapid cooling as effectively aborts discharges in in vitro epilepsy models as does slow cooling, but the magnitude of the temperature change required is less. Practical devices to inhibit seizure activity may only need to induce small temperature drops, if the cooling can be applied sufficiently rapidly.

4-Aminopyridine↗
PubMed2006

FE65 interaction with the ApoE receptor ApoEr2.

The adaptor protein FE65 interacts with the beta-amyloid precursor protein (APP) via its C-terminal phosphotyrosine binding (PTB) domain and affects APP processing and Abeta production. Our previous data demonstrate that the apoE receptor ApoEr2 co-precipitated with APP and suggest that there are extracellular and intracellular interactions between these two transmembrane proteins. We hypothesized that FE65 acts as an intracellular link between ApoEr2 and APP. Co-immunoprecipitation experiments in COS7 cells demonstrated an interaction between ApoEr2 and FE65 that depended on the N-terminal PTB domain of FE65. Full-length FE65 increased co-immunoprecipitation of ApoEr2 and APP. Full-length FE65 also increased surface expression of ApoEr2, as determined by surface protein biotinylation and live cell surface staining. Constructs containing both the C- and N-terminal PTB domains of FE65 increased secreted APP, secreted ApoEr2, APP C-terminal fragment, and ApoEr2 C-terminal fragment, but constructs containing only single PTB domains did not affect APP or ApoEr2 processing. In addition, full-length FE65 decreased Abeta to a significantly greater extent than individual FE65 domains. These data suggest that FE65 can bind APP and ApoEr2 at the same time and affect the processing of each.

Amyloid beta-Protein Precursor↗
PubMed2006

Remodeling of synaptic structures in the motor cortex following spinal cord injury.

After spinal cord injury (SCI), structural reorganization occurs at multiple levels of the motor system including the motor cortex, and this remodeling may underlie recovery of motor function. The present study determined whether SCI leads to a remodeling of synaptic structures in the motor cortex. Dendritic spines in the rat motor cortex were visualized by confocal microscopy in fixed slices, and their density and morphology were analyzed after an overhemisection injury at C4 level. Spine density decreased at 7 days and partially recovered by 28 days. Spine head diameter significantly increased in a layer-specific manner. SCI led to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10% increase in mean spine length. In addition, filopodium-like long dendritic protrusions were more frequently observed after SCI, suggesting an increase in synaptogenic events. This spine remodeling was accompanied by increased expression of polysialylated neural cell adhesion molecule, which attenuates adhesion between the pre- and postsynaptic membranes, in the motor cortex from as early as 3 days to 2 weeks after injury, suggesting a decrease in synaptic adhesion during the remodeling process. These results demonstrate time-dependent changes in spine density and morphology in the motor cortex following SCI. This synaptic remodeling seems to proceed with a time scale ranging from days to weeks. Elongation of dendritic spines may indicate a more immature and modifiable pattern of synaptic connectivity in the motor cortex being reorganized following SCI.

Amino Acids↗
PubMed2006

The pheromone androstenol (5 alpha-androst-16-en-3 alpha-ol) is a neurosteroid positive modulator of GABAA receptors.

Androstenol is a steroidal compound belonging to the group of odorous 16-androstenes, first isolated from boar testes and also found in humans. Androstenol has pheromone-like properties in both animals and humans, but the molecular targets of its pheromonal activity are unknown. Androstenol is structurally similar to endogenous A-ring reduced neurosteroids that act as positive modulators of GABA(A) receptors. Here we show that androstenol has neurosteroid-like activity as a GABA(A) receptor modulator. In whole-cell recordings from cerebellar granule cells, androstenol (but not its 3beta-epimer) caused a concentration-dependent enhancement of GABA-activated currents (EC(50), 0.4 microM in cultures; 1.4 microM in slices) and prolonged the duration of spontaneous and miniature inhibitory postsynaptic currents. Androstenol (0.1-1 microM) also potentiated the amplitude of GABA-activated currents in human embryonic kidney 293 cells transfected with recombinant alpha1beta2gamma2 and alpha2beta2gamma2 GABA(A) receptors and, at high concentrations (10-300 microM), directly activated currents in these cells. Systemic administration of androstenol (30-50 mg/kg) caused anxiolytic-like effects in mice in the open-field test and elevated zero-maze and antidepressant-like effects in the forced swim test (5-10 mg/kg). Androstenol, but not its 3beta-epimer, conferred seizure protection in the 6-Hz electroshock and pentylenetetrazol models (ED(50) values, 21.9 and 48.9 mg/kg, respectively). The various actions of androstenol in the whole-animal models are consistent with its activity as a GABA(A) receptor modulator. GABA(A) receptors could represent a target for androstenol as a pheromone, for which it is well suited because of high volatility and lipophilicity, or as a conventional hormonal neurosteroid.

Androstenols↗
PubMed2006

Phenotypic changes in NG2+ cells after spinal cord injury.

Following contusive spinal cord injury (SCI), 50% of oligodendrocytes in the residual white matter are lost within 24 h. NG2-expressing cell proliferation is maximal 3 days after SCI, and may be the source of mature oligodendrocytes and astrocytes that chronically replace those that were lost. We studied NG2(+) cells dissociated from the 3-day injured spinal cord for comparison with those from uninjured adult and early postnatal cords. After 24 h in serum-containing medium, we performed patch clamp analysis and immunocytochemistry for NG2 in combination with nestin (progenitors), and A2B5, O4, and O1 (oligodendrocyte lineage markers). We observed an NG2(+)/A2B5-/O4-/O1- population in both adult preparations. More than double the normal number of NG2(+) cells was isolated from the injured cord, but OX42(+) microglia/macrophages were the predominant cell type after injury. Most cells isolated at P7 were NG2-/A2B5(+), whereas those from the normal adult were NG2(+)/A2B5-. NG2(+) cells after SCI displayed altered voltage-gated potassium current profiles compared to normal adult and P7 animals. Additionally, less than 25% of adult cells (normal and injured) responded to GABA and glutamate, compared to 100% of P7 cells. Our results indicate that the adult NG2(+) cell pool is antigenically and physiologically different than the early postnatal pool, and that contusive injury induces changes in adult NG2(+) cells.

Animals↗
PubMed2005

Apolipoprotein E receptor 2 interactions with the N-methyl-D-aspartate receptor.

In our previous studies we showed that apoE treatment of neurons activated ERK 1/2 signaling, and activation was blocked by treatment with inhibitors of the low density lipoprotein receptor family, the N-methyl-d-aspartate (NMDA) receptor antagonist MK 801, and calcium channel blockers. We hypothesized an interaction between the low density lipoprotein receptor family members and the NMDA receptor. In the present study, we confirmed through co-immunoprecipitation experiments an interaction between the apoE receptor, ApoEr2, and NMDAR1 through their extracellular domains. We also found that the PDZ1 domain of PSD95, a postsynaptic scaffolding protein, interacted with the C terminus of ApoEr2 via an alternatively spliced, intracellular exon. This interaction between ApoEr2 and PSD95 in neurons was modulated by NMDA receptor activation and an ApoEr2 ligand. We also found that the PDZ2 domain of PSD95 interacted with the NR2A and NR2B subunits of NMDA receptors. Full-length PSD95 increased cell surface levels of ApoEr2 and its cleavage, resulting in increases in secreted ApoEr2 and C-terminal fragments of ApoEr2. These studies suggest that ApoEr2 can form a multiprotein complex with NMDA receptor subunits and PSD95.

Alternative Splicing↗
PubMed2005

The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2.

The NMDA receptor (NMDAR) is a component of excitatory synapses and a key participant in synaptic plasticity. We investigated the role of two domains in the C terminus of the NR2B subunit--the PDZ binding domain and the clathrin adaptor protein (AP-2) binding motif--in the synaptic localization of NMDA receptors. NR2B subunits lacking functional PDZ binding are excluded from the synapse. Mutations in the AP-2 binding motif, YEKL, significantly increase the number of synaptic receptors and allow the synaptic localization of NR2B subunits lacking PDZ binding. Peptides corresponding to YEKL increase the synaptic response within minutes. In contrast, the NR2A subunit localizes to the synapse in the absence of PDZ binding and is not altered by mutations in its motif corresponding to YEKL of NR2B. This study identifies a dynamic regulation of synaptic NR2B-containing NMDARs through PDZ protein-mediated stabilization and AP-2-mediated internalization that is modulated by phosphorylation by Fyn kinase.

Amino Acid Motifs↗
PubMed2005

NMDA receptors increase the size of GABAergic terminals and enhance GABA release.

In developing cerebellar interneurons, NMDA increases spontaneous GABA release by activating presynaptic NMDA receptors. We investigated the role of these receptors on differentiating basket/stellate cells in cerebellar cultures grown under conditions allowing functional synaptic transmission. Presynaptic GABAergic boutons were visualized either by GAD65 immunostaining or by using cells derived from GAD65-enhanced green fluorescent protein (eGFP) transgenic mice, in which cerebellar basket/stellate cells express eGFP. After the first week in culture, whole-cell recordings from granule cells reveal that acute application of NMDA increases miniature IPSC (mIPSC) frequency. Interestingly, after 2 weeks, the mIPSC frequency increases compared with the first week but is not modulated by NMDA. Furthermore, in cultures chronically treated with NMDA for 1 week, the size of the GABAergic boutons increases. This growth is paralleled by increased mIPSC frequency and the loss of NMDA sensitivity. Direct patch-clamp recording from these presynaptic terminals reveals single NMDA-activated channels, showing multiple conductance levels, and electronic propagation from the somatodendritic compartment. Our results demonstrate that NMDA receptors alter GABAergic synapses in developing cerebellar cultures by increasing the size of the terminal and spontaneous GABA release. These findings parallel changes in inhibitory synaptic efficacy seen in vivo in developing GABAergic interneurons of the molecular layer of the cerebellum.

Animals↗
PubMed2005

Deletion of the NR2A subunit prevents developmental changes of NMDA-mEPSCs in cultured mouse cerebellar granule neurones.

We investigated the role N-methyl-d-aspartate (NMDA) receptor subunits play in shaping excitatory synaptic currents in cultures of cerebellar granule cells (CGCs) from NR2A knockout (NR2A-/-) and wild-type (+/+) mice. Cultures were maintained in a condition that facilitates the occurrence of functional synapses, allowing us to record NMDA-miniature excitatory postsynaptic currents (mEPSCs) in addition to NMDA receptor-mediated whole-cell currents at three ages in vitro. Whole-cell NMDA current density decreased with development in both strains though currents from NR2A-/- neurones demonstrated greater sensitivity to CP101 606, an NR2B subunit specific blocker. Sensitivity to Mg(2+) blockade decreased with age in vitro in +/+ but not in NR2A-/- CGCs. Immunocytochemistry revealed that dendrites and somas displayed distinct NR1 and NR2A subunit clusters which became increasingly colocalized in +/+ neurones. Qualitatively the overall NR2B subunit staining pattern was similar in +/+ and NR2A-/- neurones throughout development, suggesting that the NR2B subunit distribution is not mediated by the NR2A subunit. In addition, staining with markers for excitatory synapses showed that expression of NR2A subunit (but not NR2B) increases at both synaptic and extrasynaptic sites in +/+ neurones during development. In parallel, NMDA-mEPSCs were faster in +/+ compared with NR2A-/- neurones at all time points studied, suggesting that the NR2A subunit begins to replace NR2B-rich NMDA receptors even at early stages of development. Many NR2A-/- neurones were devoid of NMDA-mEPSCs at the later time point, and transfection of the NR2A subunit in these neurones restored fast decay and the occurrence of NMDA-mEPSCs. Taken together, our results indicate that the NR2A subunit is mainly responsible for the developmental changes observed in the maturation of excitatory synapses.

Action Potentials↗
PubMed2005

Developmental changes of GABA synaptic transient in cerebellar granule cells.

The time course of synaptic currents is largely determined by the microscopic gating of the postsynaptic receptors and the temporal profile of the synaptic neurotransmitter concentration. Although several lines of evidence indicate that developmental changes of GABAergic synaptic current time course are clearly correlated with a switch in postsynaptic receptors, much less is known about the modification of GABA release during development. To address this issue, we studied the sensitivity of miniature inhibitory postsynaptic currents (mIPSCs) to a quickly dissociating competitive antagonist, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), in neurons cultured for 6 to 8 days in vitro (DIV) ("young") and for 12 to 14 DIV ("old"). mIPSCs recorded in young neurons were significantly more resistant to the block by TPMPA. This observation was interpreted as a consequence of a more efficient displacement of TPMPA from GABA(A) receptors caused by a stronger GABA release in young neurons. The change in mIPSC sensitivity to TPMPA during development was not affected by the deletion of alpha(1) subunit, supporting its presynaptic origin. The effects of a second quickly dissociating antagonist, SR-95103 [2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridazinium chloride], on young, old, and alpha(1) -/- neurons were qualitatively the same as those obtained with TPMPA. Moreover, the analysis of current responses to ultrafast GABA applications showed that the unbinding rates of TPMPA in DIV 6 to 8 and in DIV 12 to 14 neurons are not significantly different, ruling out the postsynaptic mechanism of differential TPMPA action. Thus, we provide evidence that presynaptic GABA uniquantal release is developmentally regulated.

Age Factors↗
PubMed2004

The nicotinic receptor in the rat pineal gland is an alpha3beta4 subtype.

The rat pineal gland contains a high density of neuronal nicotinic acetylcholine receptors (nAChRs). We characterized the pharmacology of the binding sites and function of these receptors, measured the nAChR subunit mRNA, and used subunit-specific antibodies to establish the receptor subtype as defined by subunit composition. In ligand binding studies, [3H]epibatidine ([3H]EB) binds with an affinity of approximately 100 pM to nAChRs in the pineal gland, and the density of these sites is approximately 5 times that in rat cerebral cortex. The affinities of nicotinic drugs for binding sites in the pineal gland are similar to those at alpha3beta4 nAChRs heterologously expressed in human embryonic kidney 293 cells. In functional studies, the potencies and efficacies of nicotinic drugs to activate or block whole-cell currents in dissociated pinealocytes match closely their potencies and efficacies to activate or block 86Rb+ efflux in the cells expressing heterologous alpha3beta4 nAChRs. Measurements of mRNA indicated the presence of transcripts for alpha3, beta2, and beta4 nAChR subunits but not those for alpha2, alpha4, alpha5, alpha6, alpha7, or beta3 subunits. Immunoprecipitation with subunit-specific antibodies showed that virtually all [3H]EB-labeled nAChRs contained alpha3 and beta4 subunits associated in one complex. The beta2 subunit was not associated with this complex. Taken together, these results indicate that virtually all of the nAChRs in the rat pineal gland are the alpha3beta4 nAChR subtype and that the pineal gland can therefore serve as an excellent and convenient model in which to study the pharmacology and function of these receptors in a native tissue.

Animals↗
PubMed2004

Expression of distinct alpha subunits of GABAA receptor regulates inhibitory synaptic strength.

Distinct alpha subunit subtypes in the molecular assembly of GABA(A) receptors are a critical determinant of the functional properties of inhibitory synapses and their modulation by a range of pharmacological agents. We investigated the contribution of these subunits to the developmental changes of inhibitory synapses in cerebellar granule neurons in primary cultures from wild-type and alpha1 subunit -/- mice. The decay time of miniature inhibitory postsynaptic currents (mIPSCs) halved between 6 days in vitro (DIV6) and DIV12. This was paralleled by the decrease of alpha2 and alpha3 subunits, the increase of alpha1 and alpha6 subunits expression at synapses, and changes in the action of selective alpha subunit modulators. A small but significant shortening of mIPSCs was observed with development in cells from -/- mice together with a decrease in the expression of alpha3 subunit. In contrast, the expression of alpha2 subunit at inhibitory synapses in -/- cells was significantly higher than in +/+ cells at DIV11-12. alpha5 subunit was not detected, and increased sensitivity to a selective alpha4/alpha6 subunit agonist suggests increased expression of extrasynaptic receptors in -/- mice. beta2/beta3 subunit expression and loreclezole sensitivity increased with development in +/+ but not in -/- cells, supporting the preferential association of the alpha1 with the beta2 subunit. Synaptic charge transfer strongly decreased with development but was not different between cells in the +/+ and -/- groups until DIV11-12. Our results uncover a pattern of sequential expression of alpha subunits underlying the changes in functional efficacy of GABAergic networks with development.

Animals↗
PubMed2004

Repeated electroconvulsive stimulation impairs long-term depression in the neostriatum.

BACKGROUND: Increasing and consistent findings of structural and functional abnormalities in patients with mood disorders demonstrate a clear involvement of the neostriatum. Therefore, the beneficial effect of electroconvulsive stimulation (ECS) treatment of acute state of mood disorder may relate to changes in striatal synaptic plasticity. METHODS: We studied the effect of ECS treatment on the reported long-term depression (LTD) of synaptic excitatory afferents to striatal medium spiny neurons. Stimulation of the white matter between the cortex and the striatum elicited excitatory postsynaptic potentials (EPSPs) in medium spiny neurons in rat corticostriatal slices while recording using the whole-cell patch-clamp technique. RESULTS: The EPSPs evoked in striatal neurons undergo LTD with repeated stimulation trains, and LTD of this pathway is impaired in rats after ECS treatment for 1 week, similar to what is reported in chronic lithium treatment. Electroconvulsive stimulation did not affect intrinsic membrane properties or the occurrence of spontaneous EPSCs. Dose-dependent inhibition of the EPSPs by a nonselective agonist of metabotropic glutamate receptor did not change in rats after ECS treatment. CONCLUSIONS: Our data suggest that the effectiveness of electroconvulsive therapy in mood disorders may be a consequence of LTD impairment of excitatory cortical afferents to striatal projecting neurons.

Animals↗
PubMed2004

Genetic manipulations of GABAA receptor in mice make inhibition exciting.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) plays an important role in brain development and behavior. GABA(A) receptor subunits knock-out and knock-in mice have proven that GABA(A) receptors are involved in control of motor coordination, learning, and memory and play a role in anxiety, panic, and epileptogenesis. In addition, these receptors are involved in the molecular mechanisms of action of many drugs and participate actively in cortical plasticity. The use of genetically engineered mice has perhaps never been as successful as in understanding the importance of the heterogeneity of GABA(A) receptors. We review these findings and speculate on the new directions that the use of mice with altered expression of GABA(A) receptor subunits may provide.

Animals↗