Daniela Gavello

The effect of CdSe-ZnS quantum dots on calcium currents and catecholamine secretion in mouse chromaffin cells

Semiconductor nanocrystal quantum dots (QDs) possess an enormous potential of applications in nanomedicine, drug delivery and bioimaging which derives from their unique photoemission and photostability characteristics. In spite of this, however, their interactions with biological systems and impact on human health are still largely unknown. Here we used neurosecretory mouse chromaffin cells of the adrenal gland for testing the effects of CdSe-ZnS core-shell quantum dots (5-36 nM) on Ca(2+) channels functionality and Ca(2+)-dependent neurosecretion. Prolonged exposure (24 h) to commonly used concentrations of CdSe-ZnS QDs (≥16 nM) showed that the semiconductor nanocrystal is effectively internalized into the cells without affecting cell integrity (no changes of membrane resistance and cell capacitance). QDs reduced the size of Ca(2+) currents by ∼28% in a voltage-independent manner without affecting channel gating. Correspondingly, depolarization-evoked exocytosis, measured at +10 mV, where Ca(2+) currents are maximal, was reduced by 29%. CdSe-ZnS QDs reduced the size of the readily releasable pool (RRP) of secretory vesicles by 32%, the frequency of release by 33% and the overall quantity of released catecholamines by 61%, as measured by carbon fibers amperometry. In addition, the Ca(2+)-dependence of exocytosis was reduced, whereas the catecholamine content of single granules, as well as the kinetics of release, remained unaltered. These data suggest that exposure to CdSe-ZnS QDs impairs Ca(2+) influx and severely interferes with the functionality of the exocytotic machinery, compromising the overall catecholamine supply from chromaffin cells.

Rapid neurite outgrowth in neurosecretory cells and neurons is sustained by the exocytosis of a cytoplasmic organelle, the enlargeosome

Neurite outgrowth is known as a slow (days) process occurring in nerve cells and neurons during neurotrophin treatment and upon transfer to culture, respectively. Using Y27632, a drug that induces activation of Rac1, a downstream step of the neurotrophin signaling cascade, we have identified a new form of outgrowth, which is rapid (<1 hour) and extensive (>500 μm2 surface enlargement/single cell/first hour). However, this outgrowth takes place only in cells (PC12-27 and SH-SY5Y cells, and embryonic and neonatal neurons) rich in an exocytic organelle, the enlargeosome. Golgi vesicles, TGN vesicles and endosomes are not involved. The need for enlargeosomes for plasma-membrane expansion was confirmed by the appearance of their marker, Ahnak, at the cell surface and by the dependence of neurite outgrowth on VAMP4, the vSNARE of enlargeosome exocytosis. In enlargeosome-rich cells, VAMP4 downregulation also attenuated the slow outgrowth induced by nerve growth factor (NGF). Similar to NGF-induced neurite outgrowth in enlargeosome-lacking cells, the new, rapid, Y27632-induced process required microtubules. Other properties of neurite outgrowth in cells lacking enlargeosomes — such as dependence on VAMP7, on microfilaments, on gene transcription and on protein synthesis, and blockade of mitoses and accumulation of neuronal markers — were not evident. The enlargeosome-sustained process might be useful for the rapid neurite outgrowth at peculiar stages and/or conditions of nerve and neuronal cells. However, its properties and its physiological and pathological role remain to be investigated.

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca2+-dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3-/- KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca2+ currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca2+ loading during long interspike intervals and, possibly, to control the Ca2+-dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.

Synthesis, Physicochemical Characterization, and Biological Activities of New Carnosine Derivatives Stable in Human Serum As Potential Neuroprotective Agents

The synthesis and the physicochemical and biological characterization of a series of carnosine amides bearing on the amido group alkyl substituents endowed with different lipophilicity are described. All synthesized products display carnosine-like properties differentiating from the lead for their high serum stability. They are able to complex Cu(2+) ions at physiological pH with the same stoichiometry as carnosine. The newly synthesized compounds display highly significant copper ion sequestering ability and are capable of protecting LDL from oxidation catalyzed by Cu(2+) ions, the most active compounds being the most hydrophilic ones. All the synthesized amides show quite potent carnosine-like HNE quenching activity; in particular, 7d, the member of the series selected for this kind of study, is able to cross the blood-brain barrier (BBB) and to protect primary mouse hippocampal neurons against HNE-induced death. These products can be considered metabolically stable analogues of carnosine and are worthy of additional investigation as potential neuroprotective agents.

Leptin Counteracts the Hypoxia-Induced Inhibition of Spontaneously Firing Hippocampal Neurons: A Microelectrode Array Study

Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O2). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV). Under normoxic conditions, hippocampal neurons were spontaneously firing, either with prevailing isolated and randomly distributed spikes (11 DIV), or with patterns characterized by synchronized bursts (18 DIV). Exposure to hypoxia severely impaired the spontaneous activity of hippocampal neurons, reducing their firing frequency by 54% and 69%, at 11 and 18 DIV respectively, and synchronized their firing activity. Pretreatment with 50 nM leptin reduced the firing frequency of normoxic neurons and contrasted the hypoxia-induced depressive action, either by limiting the firing frequency reduction (at both ages) or by increasing it to 126% (in younger neurons). In order to find out whether leptin exerts its effect by activating large conductance Ca2+-activated K+ channels (BK), as shown on rat hippocampal neurons, we applied the BK channel blocker paxilline (1 µM). Our data show that paxilline reversed the effects of leptin, both on normoxic and hypoxic neurons, suggesting that the adipokine counteracts hypoxia through BK channels activation in mouse hippocampal neurons.

Altered excitability of cultured chromaffin cells following exposure to multi-walled carbon nanotubes

Abstract We studied the effects of multi-walled carbon nanotubes (MWCNTs) on the electrophysiological properties of cultured mouse chromaffin cells, a model of spontaneously firing cells. The exposure of chromaffin cells to MWCNTs at increasing concentrations (30–263 μg/ml) for 24 h reduced, in a dose-dependent way, both the cell membrane input resistance and the number of spontaneously active cells (from 80–52%). Active cells that survived from the toxic effects of MWCNTs exhibited more positive resting potentials, higher firing frequencies and unaltered voltage-gated Ca2+, Na+ and K+ current amplitudes. MWCNTs slowed down the inactivation kinetics of Ca2+-dependent BK channels. These electrophysiological effects were accompanied by MWCNTs internalization, as confirmed by transmission electron microscopy, indicating that most of the toxic effects derive from a dose-dependent MWCNTs-cell interaction that damages the spontaneous cell activity.

Bud extracts from Tilia tomentosa Moench inhibit hippocampal neuronal firing through GABAA and benzodiazepine receptors activation

ETHNOPHARMACOLOGICAL RELEVANCE Tilia tomentosa Moench bud extracts (TTBEs) is used in traditional medicine for centuries as sedative compound. Different plants belonging to the Tilia genus have shown their efficacy in the treatment of anxiety but still little is known about the mechanism of action of their bud extracts. AIM OF THE STUDY To evaluate the action of TTBEs as anxiolytic and sedative compound on in vitro hippocampal neurons. MATERIAL AND METHODS The anxiolytic effect of TTBEs was assayed by testing the effects of these compounds on GABAA receptor-activated chloride current of hippocampal neurons by means of the patch-clamp technique and microelectrode-arrays (MEAs). RESULTS TTBEs acutely administered on mouse hippocampal neurons, activated a chloride current comparable to that measured in the presence of GABA (100 µM). Bicuculline (100 µM) and picrotoxin (100 µM) blocked about 90% of this current, while the remaining 10% was blocked by adding the benzodiazepine (BDZ) antagonist flumazenil (30 µM). Flumazenil alone blocked nearly 60% of the TTBEs activated current, suggesting that TTBEs binds to both GABAA and BDZ receptor sites. Application of high-doses of TTBEs on spontaneous active hippocampal neurons grown for 3 weeks on MEAs blocked the synchronous activity of these neurons. The effects were mimicked by GABA and prevented by picrotoxin (100µM) and flumazenil (30 µM). At minimal doses, TTBEs reduced the frequency of synchronized bursts and increased the cross-correlation index of synchronized neuronal firing. CONCLUSIONS Our data suggest that TTBEs mimics GABA and BDZ agonists by targeting hippocampal GABAergic synapses and inhibiting network excitability by increasing the strength of inhibitory synaptic outputs. Our results contribute toward the validation of TTBEs as effective sedative and anxiolytic compound.

Disruption of ArhGAP15 results in hyperactive Rac1, affects the architecture and function of hippocampal inhibitory neurons and causes cognitive deficits

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15−/− mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.

Leptin-mediated ion channel regulation: PI3K pathways, physiological role, and therapeutic potential

ABSTRACT Leptin is produced by adipose tissue and identified as a “satiety signal,” informing the brain when the body has consumed enough food. Specific areas of the hypothalamus express leptin receptors (LEPRs) and are the primary site of leptin action for body weight regulation. In response to leptin, appetite is suppressed and energy expenditure allowed. Beside this hypothalamic action, leptin targets other brain areas in addition to neuroendocrine cells. LEPRs are expressed also in the hippocampus, neocortex, cerebellum, substantia nigra, pancreatic β-cells, and chromaffin cells of the adrenal gland. It is intriguing how leptin is able to activate different ionic conductances, thus affecting excitability, synaptic plasticity and neurotransmitter release, depending on the target cell. Most of the intracellular pathways activated by leptin and directed to ion channels involve PI3K, which in turn phosphorylates different downstream substrates, although parallel pathways involve AMPK and MAPK. In this review we will describe the effects of leptin on BK, KATP, KV, CaV, TRPC, NMDAR and AMPAR channels and clarify the landscape of pathways involved. Given the ability of leptin to influence neuronal excitability and synaptic plasticity by modulating ion channels activity, we also provide a short overview of the growing potentiality of leptin as therapeutic agent for treating neurological disorders.

Dual action of leptin on rest-firing and stimulated catecholamine release via phosphoinositide 3-kinase-driven BK channel up-regulation in mouse chromaffin cells

Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite‐suppressing effect and, as such, exerts a relevant action on the adipo‐adrenal axis. Leptin has a dual action on adrenal mouse chromaffin cells both at rest and during stimulation. At rest, the adipokine inhibits the spontaneous firing of most cells by enhancing the probability of BK channel opening through the phosphoinositide 3‐kinase signalling cascade. This inhibitory effect is absent in db–/db– mice deprived of Ob receptors. During sustained stimulation, leptin preserves cell excitability by generating well‐adapted action potential (AP) trains of lower frequency and broader width and increases catecholamine secretion by increasing the size of the ready‐releasable pool and the rate of vesicle release. In conclusion, leptin dampens AP firing at rest but preserves AP firing and enhances catecholamine release during sustained stimulation, highlighting the importance of the adipo‐adrenal axis in the leptin‐mediated increase of sympathetic tone and catecholamine release.