Giulio Sancini

Ionic mechanisms underlying burst firing in pyramidal neurons: intracellular study in rat sensorimotor cortex

In in vitro slices prepared from rat sensorimotor cortex, intracellular recordings were obtained from 107 layer V pyramidal neurons, subsequently injected with biocytin for morphological reconstruction. Of the 107 neurons, 59 (55.1%) were identified as adapting (45) or non-adapting (13) regular spiking neurons (RS), and 48 (44.9%) as intrinsically bursting (IB) neurons discharging with an initial cluster of action potentials, which tended to recur rhythmically in a subset of 19 cells. The block of IAR by extracellular Cs+ did not affect burst generation, but enhanced the tendency to reburst in IB neurons. A similar effect was induced by other procedures affecting K(+)-dependent post-burst hyperpolarization. In IB neurons Ca2+ spikes had a longer decay time than in RS neurons, however selective blockers of both low and high threshold Ca2+ conductances failed to impair bursting activity. On the contrary, the perfusion of the slices with 0.5-1 microM TTX suppressed bursting behaviour in a critical time interval preceding the complete block of Na(+)-dependent action potentials. It is concluded that the persistent Na+ current INAP is the most important intrinsic factor for the typical firing properties of IB neurons, while Ca2+ and K+ conductances appear to contribute towards shaping bursts and controlling their recurrence rate. The morphology, connectivity and physiological properties of adapting and non-adapting RS neurons are particularly suited to the processing of respectively phasic and tonic inputs, whereas the properties of IB neurons are consistent with their suggested role in cortical rhythmogenesis and in the pathophysiological synchronized activities underlying epileptogenesis.

Translocation pathways for inhaled asbestos fibers

We discuss the translocation of inhaled asbestos fibers based on pulmonary and pleuro-pulmonary interstitial fluid dynamics. Fibers can pass the alveolar barrier and reach the lung interstitium via the paracellular route down a mass water flow due to combined osmotic (active Na+ absorption) and hydraulic (interstitial pressure is subatmospheric) pressure gradient. Fibers can be dragged from the lung interstitium by pulmonary lymph flow (primary translocation) wherefrom they can reach the blood stream and subsequently distribute to the whole body (secondary translocation). Primary translocation across the visceral pleura and towards pulmonary capillaries may also occur if the asbestos-induced lung inflammation increases pulmonary interstitial pressure so as to reverse the trans-mesothelial and trans-endothelial pressure gradients. Secondary translocation to the pleural space may occur via the physiological route of pleural fluid formation across the parietal pleura; fibers accumulation in parietal pleura stomata (black spots) reflects the role of parietal lymphatics in draining pleural fluid. Asbestos fibers are found in all organs of subjects either occupationally exposed or not exposed to asbestos. Fibers concentration correlates with specific conditions of interstitial fluid dynamics, in line with the notion that in all organs microvascular filtration occurs from capillaries to the extravascular spaces. Concentration is high in the kidney (reflecting high perfusion pressure and flow) and in the liver (reflecting high microvascular permeability) while it is relatively low in the brain (due to low permeability of blood-brain barrier). Ultrafine fibers (length < 5 μm, diameter < 0.25 μm) can travel larger distances due to low steric hindrance (in mesothelioma about 90% of fibers are ultrafine). Fibers translocation is a slow process developing over decades of life: it is aided by high biopersistence, by inflammation-induced increase in permeability, by low steric hindrance and by fibers motion pattern at low Reynolds numbers; it is hindered by fibrosis that increases interstitial flow resistances.

Prenatal methylazoxymethanol treatment in rats produces brain abnormalities with morphological similarities to human developmental brain dysgeneses

A double methylazoxymethanol (MAM) intraperitoneal injection was prenatally administered to pregnant rats at gestational day 15 to induce developmental brain dysgeneses. Thirty adult rats from 8 different progenies were investigated with a combined electrophysiological and neuroanatomical analysis. The offspring of treated dams was characterized by extensive cortical layering abnormalities, subpial bands of heterotopic neurons in layer I, and subcortical nodules of heterotopic neurons extending from the periventricular region to the hippocampus and neocortex. The phenotype of cell subpopulations within the heterotopic structures was analyzed by means of antibodies raised against glial and neuronal markers, calcium binding proteins, GABA, and AMPA glutamate receptors. Neurons within the subcortical heterotopic nodules were characterized by abnormal firing properties, with sustained repetitive bursts of action potentials. The subcortical nodules were surrounded by cell clusters with ultrastructural features of young migrating neurons. The immunocytochemical data suggested, moreover, that the subcortical heterotopia were formed by neurons originally committed to the neocortex and characterized by morphological features similar to those found in human periventricular nodular heterotopia. The present study demonstrates that double MAM treatment at gestational day 15 induces in rats developmental brain abnormalities whose anatomical and physiological features bear resemblance to those observed in human brain dysgeneses associated with intractable epilepsy. Therefore, MAM treated rats could be considered as useful tools in investigating the pathogenic mechanisms involved in human developmental brain dysgeneses.

Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model

A promising strategy to enhance blood-brain barrier penetration by drugs is the functionalization of nanocarriers with uptake-facilitating ligands. We studied the cellular uptake, by cultured RBE4 brain capillary endothelial cells, of nanoliposomes (NLs) covalently coupled with monomer or tandem dimer of apolipoprotein E (ApoE)-derived peptides (residues 141-150), at various densities. NLs without functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy and quantified by fluorescence-activated cell sorting. Functionalization with peptides mediated an efficient NLs uptake that increased with peptide density; NLs carrying monomeric peptide performed the best. Moreover, we studied the ability of ApoE-NLs to enhance the transport of a drug payload through a RBE4 cell monolayer. The permeability of a tritiated curcumin derivative was enhanced after its entrapment into ApoE-NLs, in particular those functionalized with the dimer (+83% with respect to free drug, P < 0.01). Thus, these NLs appear particularly suitable for implementing further strategies for drug brain targeting.

Postnatal differentiation of firing properties and morphological characteristics in layer V pyramidal neurons of the sensorimotor cortex

The maturational profile of the firing characteristics of 217 layer V pyramidal neurons of rat sensorimotor cortex, injected with biocytin for morphological reconstruction, was analysed by means of intracellular recordings made between postnatal day (P)3 and 22. Starting from the onset of the second postnatal week, the pyramidal neurons could be differentiated as adapting or non-adapting regular spiking on the basis of the presence or absence of spike frequency adaptation. The percentage of non-adapting regular spiking neurons was very high during the second postnatal week (53%) and progressively decreased with age, concurrently with the appearance of the new class of intrinsically bursting neurons (beginning of the third week) whose percentage progressively increased from 23%, found in P14-P16 rats, to 46% in adult rats. Non-adapting regular spiking neurons were found to share with intrinsically bursting neurons several physiological characteristics comprehending faster action potentials, more prominent effect of anomalous rectification and consistent depolarizing afterpotentials, that differentiated them from the adapting regular spiking neurons. Moreover, intrinsically bursting and non-adapting regular spiking neurons were characterized by a round-shaped distribution of basal dendrites and expanded apical dendritic arborization, that differentiated them from the adapting regular spiking neurons showing a simpler dendritic arborization. These morphological hallmarks were seen in immature intrinsically bursting neurons as soon as they became distinguishable, and in immature non-adapting regular spiking neurons starting from the onset of the second postnatal week. These findings suggest that a significant subpopulation of immature non-adapting regular spiking neurons are committed to becoming bursters, and that they are converted into intrinsically bursting neurons during the second postnatal week, as soon as the ionic current sustaining the burst firing is sufficiently strong. The faster action potentials in both immature non-adapting regular spiking and intrinsically bursting neurons suggest a higher density of Na+ channels in these neuronal classes: the maturational increase in Na+-current, namely of its persistent fraction, may represent the critical event for the conversion of the non-adapting regular spiking neurons into the intrinsically bursting ones.

Altered connections between neocortical and heterotopic areas in methylazoxymethanol-treated rat

We are currently investigating various treatments which could determine, in the rat brain, structural abnormalities mimicking those reported in human brain dysgeneses. We can induce the formation of neuronal heterotopia in the progeny of rats by means of a double injection of the cytotoxic agent methylazoxymethanol acetate (MAM) on embryonic day 15. We have now investigated the anatomical connections of these heterotopia by means of anterograde and retrograde tract tracing techniques. The induced heterotopia along the border of the lateral ventricles shared common anatomical features with the periventricular nodules in human periventricular or subcortical nodular heterotopia (PNH). The tract tracing data demonstrated the existence of reciprocal connections between the neuronal heterotopia and the ipsilateral and contralateral cortical areas, and the presence of abnormal cortico-hippocampal and cortico-cortical connections. On the basis of the connectivity patterns, it may be speculated that some cells in the heterotopia could be neurons originally committed to the cortex, that were interrupted in their migration by the MAM treatment. Given the common morphological features seen in human PNH and MAM-induced brain heterotopia, the anatomical and developmental analysis of MAM-treated rats may shed light on the mechanisms by which human brain dysgeneses develop in human patients.

Functionalization with ApoE-derived peptides enhances the interaction with brain capillary endothelial cells of nanoliposomes binding amyloid-beta peptide

Nanoliposomes containing phosphatidic acid or cardiolipin are able to target in vitro with very high affinity amyloid-β (Aβ), a peptide whose overproduction and progressive aggregation in the brain play a central role in the pathogenesis of Alzheimers disease. However, the presence of the blood-brain barrier (BBB) severely limits the penetration of either drugs or drug vehicles (nanoparticles) to the brain. Therefore, there is a need to develop and design approaches specifically driving nanoparticles to brain in a better and effective way. The aim of the present investigation is the search of a strategy promoting the interaction of liposomes containing acidic phospholipids with brain capillary endothelial cells, as a first step toward their passage across the BBB. We describe the preparation and physical characterization of nano-sized liposomes decorated with peptides derived from apolipoprotein E and characterize their interaction with human immortalized brain capillary cells cultured in vitro (hCMEC/D3). For this purpose, we synthesized two ApoE-derived peptides (the fragment 141-150 or its tandem dimer) containing a cysteine residue at the C-terminus and decorated NL by exploiting the cysteine reaction with a maleimide-group on the nanoparticle surface. NL without ApoE functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy using fluorescently labeled nanoliposomes or quantifying the cell-associated radioactivity of isotopically labeled nanoliposomes. The uptake of nanoliposomes by cell monolayers was enhanced by ApoE-peptide-functionalization, and was higher with the fragment 141-150 than with its tandem dimer. The best performance was displayed by nanoliposomes containing phosphatidic acid and decorated with the ApoE fragment 141-150. Moreover, we show that the functionalization of liposomes containing acidic phospholipids with the ApoE fragment 141-150 scarcely affects their reported ability to bind Aβ peptide in vitro. These are important and promising features for the possibility to use these nanoliposomes for the targeting of Aβ in the brain districts.

Multifunctional Liposomes Reduce Brain β-Amyloid Burden and Ameliorate Memory Impairment in Alzheimer's Disease Mouse Models

Alzheimers disease is characterized by the accumulation and deposition of plaques of β-amyloid (Aβ) peptide in the brain. Given its pivotal role, new therapies targeting Aβ are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aβ assemblies and evaluated their efficiency in reducing the Aβ burden in Alzheimers disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood–brain barrier targeting and with phosphatidic acid for Aβ binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aβ assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aβ1–42 (−33%), assessed by ELISA, and the number and total area of plaques (−34%) detected histologically. Also, brain Aβ oligomers were reduced (−70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [11C]Pittsburgh compound B (PIB). The reduction of brain Aβ was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aβ aggregates and promote peptide removal across the blood–brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimers disease.

Liposomes functionalized to overcome the blood–brain barrier and to target amyloid-β peptide: the chemical design affects the permeability across an in vitro model

Purpose We investigated the ability of amyloid-β-targeting liposomes, decorated with an anti-transferrin receptor antibody, to cross the blood–brain barrier (BBB), comparing two antibody ligation techniques. Methods Fluorescent or radiolabeled liposomes composed of sphingomyelin/cholesterol and containing phosphatidic acid, known to bind amyloid-β, were further functionalized with the anti-transferrin receptor antibody RI7217. Two different techniques were used to attach RI7217 to the liposomes surface: biotin/streptavidin linkage or thiol–maleimide covalent ligation. Surface plasmon resonance (SPR) and immunoblotting were employed to assess the nanoparticles’ binding performances. Confocal microscopy and radiochemical techniques were used for uptake and permeability studies on an in vitro BBB model made of human brain capillary endothelial cells hCMEC/D3. Results Immunoblotting experiments showed that RI7217-functionalized liposomes bind to transferrin receptor independently of the procedure employed to ligate their surface with the antibody, while SPR experiments showed a slightly higher affinity for covalently functionalized nanoliposomes. The functionalization with RI7217 did not affect the liposomes’ affinity for amyloid-β. The functionalization of liposomes with RI7217, independently of the ligation procedure, gave higher values of uptake and permeability across the barrier model in comparison to the nondecorated ones, without cell monolayer alterations. Of note, the best performing particles were those covalently coupled with the antibody. The ratios of the two radiolabeled lipids (3H-sphingomyelin and 14C-phosphatidic acid) present in the liposome bilayer were found to be similar in the apical and in the basolateral compartments of the barrier model, suggesting that liposomes were transported intact across the cell monolayer. Confocal experiments showed no co-localization of RI7217-liposomes with early/late endosomes or early lysosomes. Conclusion Our results suggest that RI7217 promotes the in vitro barrier crossing of liposomes containing phosphatidic acid, targeting the Alzheimer’s disease amyloid-β peptide. Moreover, for the first time, we prove herein the superior efficiency of covalent coupling of RI7217 versus biotin/streptavidin ligation to facilitate liposomes in overcoming the BBB in vitro.

Comparative acute lung inflammation induced by atmospheric PM and size-fractionated tire particles.

A comparison of the effects produced by size-fractionated tire particles (TP10 and TP2.5) and similar-sized urban particulate matter (PM10 and PM2.5), collected in Milan in 2007, on the lungs of mice has been performed. The focus is on early acute lung responses following intratracheal instillation of aerosolized particles at a 3-h recovery period. Together with bronchoalveolar lavage (BAL) conventional endpoints like total and differential cell counts, total protein, alkaline phosphatase, lactate dehydrogenase and pro-inflammatory cytokines (TNF-alpha, MIP-2), the expression of different stress protein markers (caspase8, Hsp70, H0-1, NF-kB) was evaluated 3h after particle instillation into Balb/c mice. The TP2.5 fraction reached the alveolar spaces and produced an acute inflammatory response as evidenced by increased LDH and AP activities, total protein and Hsp70 content. TNF-alpha and MIP-2 production was significantly increased and polymorphonuclear neutrophils (PMN) recruitment was apparent. The TP10 fraction distributed mainly in the bronchial district and the only modified BAL parameter was the expression of MIP-2. PM2.5 induced an inflammatory response lesser in magnitude than that produced by PM10 fraction. The TNF-alpha increase was not significant, and HO-1, though significantly increased with respect to the control, was unable to reduce NF-kB activation, suggesting a role of the endotoxin component of PM in stimulating a pro-inflammatory limited response. This response was maximized by the PM10 that induced a significant increase in MIP-2, TNF-alpha, and HO-1. Lung immunohistochemistry showed fine particles, TPs in particular, being able to deeply penetrate and rapidly induce inflammatory events in the parenchyma, even involving endothelial cells, while PM10 produced a strong pro-inflammatory response mediated by the bronchiolar cells and residential macrophages of the proximal alveolar sacs, likely as a consequence of its larger dimension and endotoxin content. These results provide evidence of variable inflammatory mechanisms in mouse lungs in response to both urban PM and tire particles.