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.

We studied the effect of Amyloid β 1-42 oligomers (Abeta42) on Ca2+ dependent excitability profile of hippocampal neurons. Abeta42 is one of the Amyloid beta peptides produced by the proteolytic processing of the amyloid precursor protein and participates in the initiating event triggering the progressive dismantling of synapses and neuronal circuits. Our experiments on cultured hippocampal network reveal that Abeta42 increases intracellular Ca2+ concentration by 46% and inhibits firing discharge by 19%. More precisely, Abeta42 differently regulates ryanodine (RyRs), NMDA receptors (NMDARs), and voltage gated calcium channels (VGCCs) by increasing Ca2+ release through RyRs and inhibiting Ca2+ influx through NMDARs and VGCCs. The overall increased intracellular Ca2+ concentration causes stimulation of K+ current carried by big conductance Ca2+ activated potassium (BK) channels and hippocampal network firing inhibition. We conclude that Abeta42 alters neuronal function by means of at least 4 main targets: RyRs, NMDARs, VGCCs, and BK channels. The development of selective modulators of these channels may in turn be useful for developing effective therapies that could enhance the quality of life of AD patients during the early onset of the pathology.

SummaryNeurite 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 mm 2 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 enlargeosomelacking 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.

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.

a b s t r a c tSemiconductor 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 CdSeeZnS coreeshell quantum dots (5e36 nM) on Ca 2þ channels functionality and Ca 2þ -dependent neurosecretion. Prolonged exposure (24 h) to commonly used concentrations of CdSeeZnS 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 2þ currents by w28% in a voltage-independent manner without affecting channel gating. Correspondingly, depolarization-evoked exocytosis, measured at þ10 mV, where Ca 2þ currents are maximal, was reduced by 29%. CdSeeZnS 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 2þ -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 CdSeeZnS QDs impairs Ca 2þ influx and severely interferes with the functionality of the exocytotic machinery, compromising the overall catecholamine supply from chromaffin cells.