Developing Iron Nanochelating Agents: Preliminary Investigation of Effectiveness and Safety for Central Nervous System Applications.

Excessive iron levels are believed to contribute to the development of neurodegenerative disorders by promoting oxidative stress and harmful protein clustering. Novel chelation treatments that can effectively remove excess iron while minimizing negative effects on the nervous system are being explored. This study focuses on the creation and evaluation of innovative nanobubble (NB) formulations, shelled with various polymers such as glycol-chitosan (GC) and glycol-chitosan conjugated with deferoxamine (DFO), to enhance their ability to bind iron. Various methods were used to evaluate their physical and chemical properties, chelation capacity in diverse iron solutions and impact on reactive oxygen species (ROS). Notably, the GC-DFO NBs demonstrated the ability to decrease amyloid-ß protein misfolding caused by iron. To assess potential toxicity, in vitro cytotoxicity testing was conducted using organotypic brain cultures from the substantia nigra, revealing no adverse effects at appropriate concentrations. Additionally, the impact of NBs on spontaneous electrical signaling in hippocampal neurons was examined. Our findings suggest a novel nanochelation approach utilizing DFO-conjugated NBs for the removal of excess iron in cerebral regions, potentially preventing neurotoxic effects.

Two firing modes and well-resolved Na+, K+, and Ca2+ currents at the cell-microelectrode junction of spontaneously active rat chromaffin cell on MEAs.

We recorded spontaneous extracellular action potentials (eAPs) from rat chromaffin cells (CCs) at 37 °C using microelectrode arrays (MEAs) and compared them with intracellularly recorded APs (iAPs) through conventional patch clamp recordings at 22 °C. We show the existence of two distinct firing modes on MEAs: a ~ 4 Hz irregular continuous firing and a frequent intermittent firing mode where periods of high-intraburst frequency (~ 8 Hz) of ~ 7 s duration are interrupted by silent periods of ~ 12 s. eAPs occurred either as negative- or positive-going signals depending on the contact between cell and microelectrode: either predominantly controlled by junction-membrane ion channels (negative-going) or capacitive/ohmic coupling (positive-going). Negative-going eAPs were found to represent the trajectory of the Na+, Ca2+, and K+ currents passing through the cell area in tight contact with the microelectrode during an AP (point-contact junction). The inward Nav component of eAPs was blocked by TTX in a dose-dependent manner (IC50 ~ 10 nM) while the outward component was strongly attenuated by the BK channel blocker paxilline (200 nM) or TEA (5 mM). The SK channel blocker apamin (200 nM) had no effect on eAPs. Inward Nav and Cav currents were well-resolved after block of Kv and BK channels or in cells showing no evident outward K+ currents. Unexpectedly, on the same type of cells, we could also resolve inward L-type currents after adding nifedipine (3 µM). In conclusion, MEAs provide a direct way to record different firing modes of rat CCs and to estimate the Na+, Ca2+, and K+ currents that sustain cell firing and spontaneous catecholamines secretion.

The ryanodine receptor-calstabin interaction stabilizer S107 protects hippocampal neurons from GABAergic synaptic alterations induced by Abeta42 oligomers.

The oligomeric form of the peptide amyloid beta 42 (Abeta42) contributes to the development of synaptic abnormalities and cognitive impairments associated with Alzheimer's disease (AD). To date, there is a gap in knowledge regarding how Abeta42 alters the elementary parameters of GABAergic synaptic function. Here we found that Abeta42 increased the frequency and amplitude of miniature GABAergic currents as well as the amplitude of evoked inhibitory postsynaptic currents. When we focused on paired pulse depression (PPD) to establish whether GABA release probability was affected by Abeta42, we did not observe any significant change. On the other hand, a more detailed investigation of the presynaptic effects induced by Abeta42 by means of multiple probability fluctuation analysis and cumulative amplitude analysis showed an increase in both the size of the readily releasable pool responsible for synchronous release and the number of release sites. We further explored whether ryanodine receptors (RyRs) contributed to exacerbating these changes by stabilizing the interaction between RyRs and the accessory protein calstabin. We observed that the RyR-calstabin interaction stabilizer S107 restored the synaptic parameters to values comparable to those measured in control conditions. In conclusion, our results clarify the mechanisms of potentiation of GABAergic synapses induced by Abeta42. We further suggest that RyRs are involved in the control of synaptic activity during the early stage of AD onset and that their stabilization could represent a new therapeutical approach for AD treatment. KEY POINTS: Accumulation of the peptide amyloid beta 42 (Abeta42) is a key characteristic of Alzheimer's disease (AD) and causes synaptic dysfunctions. To date, the effects of Abeta42 accumulation on GABAergic synapses are poorly understood. The findings reported here suggest that, similarly to what is observed on glutamatergic synapses, Abeta42 modifies GABAergic synapses by targeting ryanodine receptors and causing calcium dysregulation. The GABAergic impairments can be restored by the ryanodine receptor-calstabin interaction stabilizer S107. Based on this research, RyRs stabilization may represent a novel pharmaceutical strategy for preventing or delaying AD.

Advanced real-time recordings of neuronal activity with tailored patch pipettes, diamond multi-electrode arrays and electrochromic voltage-sensitive dyes.

To understand the working principles of the nervous system is key to figure out its electrical activity and how this activity spreads along the neuronal network. It is therefore crucial to develop advanced techniques aimed to record in real time the electrical activity, from compartments of single neurons to populations of neurons, to understand how higher functions emerge from coordinated activity. To record from single neurons, a technique will be presented to fabricate patch pipettes able to seal on any membrane with a single glass type and whose shanks can be widened as desired. This dramatically reduces access resistance during whole-cell recording allowing fast intracellular and, if required, extracellular perfusion. To simultaneously record from many neurons, biocompatible probes will be described employing multi-electrodes made with novel technologies, based on diamond substrates. These probes also allow to synchronously record exocytosis and neuronal excitability and to stimulate neurons. Finally, to achieve even higher spatial resolution, it will be shown how voltage imaging, employing fast voltage-sensitive dyes and two-photon microscopy, is able to sample voltage oscillations in the brain spatially resolved and voltage changes in dendrites of single neurons at millisecond and micrometre resolution in awake animals.

Triggering Neurotransmitters Secretion from Single Cells by X-ray Nanobeam Irradiation.

The employment of ionizing radiation is a powerful tool in cancer therapy, but beyond targeted effects, many studies have highlighted the relevance of its off-target consequences. An exhaustive understanding of the mechanisms underlying these effects is still missing, and no real-time data about signals released by cells during irradiation are presently available. We employed a synchrotron X-ray nanobeam to perform the first real-time simultaneous measurement of both X-ray irradiation and in vitro neurotransmitter release from individual adrenal phaeochromocytoma (PC12) cells plated over a diamond-based multielectrode array. We have demonstrated that, in specific conditions, X-rays can alter cell activity by promoting dopamine exocytosis, and such an effect is potentially very attractive for a more effective treatment of tumors.

Amyloid Beta42 oligomers up-regulate the excitatory synapses by potentiating presynaptic release while impairing postsynaptic NMDA receptors.

KEY POINTS: NMDA receptors (NMDARs) are key molecules for controlling neuronal plasticity, learning and memory processes. Their function is impaired during Alzheimer's disease (AD) but the exact consequence on synaptic function is not yet fully identified. An important hallmark of AD onset is represented by the neuronal accumulation of Amyloid Beta42 oligomers (Abeta42) that we have recently shown to be responsible for the increased intracellular Ca2+ concentration through ryanodine receptors (RyRs). Here we characterized the effects of Abeta42 on NMDA synapses showing specific pre- and post-synaptic functional changes that lead to a potentiation of basal and synchronous NMDA synaptic transmission. These overall effects can be abolished by decreasing Ca2+ release from RyRs with specific inhibitors that we propose as new pharmacological tools for AD treatment. ABSTRACT: We have recently shown that Amyloid Beta42 oligomers (Abeta42) cause calcium dysregulation in hippocampal neurons by stimulating Ca2+ release from ryanodine receptors (RyRs) and inhibiting Ca2+ entry through NMDA receptors (NMDARs). Here, we found that Abeta42 decrease the average NMDA-activated inward current and that Ca2+ entry through NMDARs is accompanied by Ca2+ release from the stores. The overall amount of intraellular Ca2+ concentration([Ca2+ ]i ) increase during NMDA application is 50% associated with RyR opening and 50% with NMDARs activation. Addition of Abeta42 does not change this proportion. We estimated the number of NMDARs expressed in hippocampal neurons and their unitary current. We found that Abeta42 decrease the number of NMDARs without altering their unitary current. Paradoxically, the oligomer increases the size of electrically evoked eEPSCs induced by NMDARs activation. We found that this is the consequence of the increased release probability (p) of glutamate and the number of release sites (N) of NMDA synapses, while the quantal size (q) is significantly decreased as expected from the decreased number of NMDARs. An increased number of release sites induced by Abeta42 is also supported by the increased size of the ready releasable pool (RRPsyn) and by the enhanced percentage of paired pulse depression (PPD). Interestingly, the RyRs inhibitor dantrolene prevents the increase of PPD induced by Abeta42 oligomers. In conclusion, Abeta42 up-regulates NMDA synaptic responses with a mechanism involving RyRs that occurs during the early stages of Alzheimer's disease (AD) onset. This suggests that new selective modulators of RyRs may be useful for designing effective therapies to treat AD patients.

p140Cap Regulates GABAergic Synaptogenesis and Development of Hippocampal Inhibitory Circuits.

The neuronal scaffold protein p140Cap was investigated during hippocampal network formation. p140Cap is present in presynaptic GABAergic terminals and its genetic depletion results in a marked alteration of inhibitory synaptic activity. p140Cap-/- cultured neurons display higher frequency of miniature inhibitory postsynaptic currents (mIPSCs) with no changes of their mean amplitude. Consistent with a potential presynaptic alteration of basal GABA release, p140Cap-/- neurons exhibit a larger synaptic vesicle readily releasable pool, without any variation of single GABAA receptor unitary currents and number of postsynaptic channels. Furthermore, p140Cap-/- neurons show a premature and enhanced network synchronization and appear more susceptible to 4-aminopyridine-induced seizures in vitro and to kainate-induced seizures in vivo. The hippocampus of p140Cap-/- mice showed a significant increase in the number of both inhibitory synapses and of parvalbumin- and somatostatin-expressing interneurons. Specific deletion of p140Cap in forebrain interneurons resulted in increased susceptibility to in vitro epileptic events and increased inhibitory synaptogenesis, comparable to those observed in p140Cap-/- mice. Altogether, our data demonstrate that p140Cap finely tunes inhibitory synaptogenesis and GABAergic neurotransmission, thus regulating the establishment and maintenance of the proper hippocampal excitatory/inhibitory balance.

Early Alterations of Hippocampal Neuronal Firing Induced by Abeta42.

We studied the effect of Amyloid ß 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.

Electrochemical measurement of quantal exocytosis using microchips.

Carbon-fiber electrodes (CFEs) are the gold standard for quantifying the release of oxidizable neurotransmitters from single vesicles and single cells. Over the last 15 years, microfabricated devices have emerged as alternatives to CFEs that offer the possibility of higher throughput, subcellular spatial resolution of exocytosis, and integration with other techniques for probing exocytosis including microfluidic cell handling and solution exchange, optical imaging and stimulation, and electrophysiological recording and stimulation. Here we review progress in developing electrochemical electrode devices capable of resolving quantal exocytosis that are fabricated using photolithography.

Low pHo boosts burst firing and catecholamine release by blocking TASK-1 and BK channels while preserving Cav1 channels in mouse chromaffin cells.

KEY POINTS: Mouse chromaffin cells (MCCs) generate spontaneous burst-firing that causes large increases of Ca2+ -dependent catecholamine release, and is thus a key mechanism for regulating the functions of MCCs. With the aim to uncover a physiological role for burst-firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo ) from 7.4 to 6.6 induces cell depolarizations of 10-15 mV that generate bursts of ∼330 ms at 1-2 Hz and a 7.4-fold increase of cumulative catecholamine-release. Burst-firing originates from the inhibition of the pH-sensitive TASK-1-channels and a 60% reduction of BK-channel conductance at pHo 6.6. Blockers of the two channels (A1899 and paxilline) mimic the effects of pHo 6.6, and this is reverted by the Cav1 channel blocker nifedipine. MCCs act as pH-sensors. At low pHo , they depolarize, undergo burst-firing and increase catecholamine-secretion, generating an effective physiological response that may compensate for the acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue. ABSTRACT: Mouse chromaffin cells (MCCs) generate action potential (AP) firing that regulates the Ca2+ -dependent release of catecholamines (CAs). Recent findings indicate that MCCs possess a variety of spontaneous firing modes that span from the common 'tonic-irregular' to the less frequent 'burst' firing. This latter is evident in a small fraction of MCCs but occurs regularly when Nav1.3/1.7 channels are made less available or when the Slo1ß2-subunit responsible for BK channel inactivation is deleted. Burst firing causes large increases of Ca2+ -entry and potentiates CA release by ∼3.5-fold and thus may be a key mechanism for regulating MCC function. With the aim to uncover a physiological role for burst-firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo ) from 7.4 to 7.0 and 6.6 induces cell depolarizations of 10-15 mV that generate repeated bursts. Bursts at pHo 6.6 lasted ∼330 ms, occurred at 1-2 Hz and caused an ∼7-fold increase of CA cumulative release. Burst firing originates from the inhibition of the pH-sensitive TASK-1/TASK-3 channels and from a 40% BK channel conductance reduction at pHo 7.0. The same pHo had little or no effect on Nav, Cav, Kv and SK channels that support AP firing in MCCs. Burst firing of pHo 6.6 could be mimicked by mixtures of the TASK-1 blocker A1899 (300 nm) and BK blocker paxilline (300 nm) and could be prevented by blocking L-type channels by adding 3 µm nifedipine. Mixtures of the two blockers raised cumulative CA-secretion even more than low pHo (∼12-fold), showing that the action of protons on vesicle release is mainly a result of the ionic conductance changes that increase Ca2+ -entry during bursts. Our data provide direct evidence suggesting that MCCs respond to low pHo with sustained depolarization, burst firing and enhanced CA-secretion, thus mimicking the physiological response of CCs to acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue.

Microelectrode Arrays of Diamond-Insulated Graphitic Channels for Real-Time Detection of Exocytotic Events from Cultured Chromaffin Cells and Slices of Adrenal Glands.

A microstructured graphitic 4 × 4 multielectrode array was embedded in a single-crystal diamond substrate (4 × 4 µG-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time-effective fabrication of extended arrays with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20 × 3.5 µm(2)) separated by 200 µm gaps. Taking advantage of the array geometry we addressed the following specific issues: (i) detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, (ii) resolve the waveform of different subsets of exocytotic events, and (iii) monitoring quantal secretory events from thin slices of the adrenal gland. The frequency of spontaneous release was low (0.12 and 0.3 Hz, respectively, for adrenal slices and cultured cells) and increased up to 0.9 Hz after stimulation with 30 mM KCl in cultured cells. The spike amplitude as well as rise and decay time were comparable with those measured by carbon fiber microelectrodes and allowed to identify three different subsets of secretory events associated with "full fusion" events, "kiss-and-run" and "kiss-and-stay" exocytosis, confirming that the device has adequate sensitivity and time resolution for real-time recordings. The device offers the significant advantage of shortening the time to collect data by allowing simultaneous recordings from cell populations either in primary cell cultures or in intact tissues.

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

KEY POINTS: 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. ABSTRACT: Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect. Besides being expressed in the hypothalamus and hippocampus, leptin receptors (ObRs) are also present in chromaffin cells of the adrenal medulla. In the present study, we report the effect of leptin on mouse chromaffin cell (MCC) functionality, focusing on cell excitability and catecholamine secretion. Acute application of leptin (1 nm) on spontaneously firing MCCs caused a slowly developing membrane hyperpolarization followed by complete blockade of action potential (AP) firing. This inhibitory effect at rest was abolished by the BK channel blocker paxilline (1 µm), suggesting the involvement of BK potassium channels. Single-channel recordings in 'perforated microvesicles' confirmed that leptin increased BK channel open probability without altering its unitary conductance. BK channel up-regulation was associated with the phosphoinositide 3-kinase (PI3K) signalling cascade because the PI3K specific inhibitor wortmannin (100 nm) fully prevented BK current increase. We also tested the effect of leptin on evoked AP firing and Ca(2+) -driven exocytosis. Although leptin preserves well-adapted AP trains of lower frequency, APs are broader and depolarization-evoked exocytosis is increased as a result of the larger size of the ready-releasable pool and higher frequency of vesicle release. The kinetics and quantal size of single secretory events remained unaltered. Leptin had no effect on firing and secretion in db(-) /db(-) mice lacking the ObR gene, confirming its specificity. In conclusion, leptin exhibits a dual action on MCC activity. It dampens AP firing at rest but preserves AP firing and increases catecholamine secretion during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release.

Reduced availability of voltage-gated sodium channels by depolarization or blockade by tetrodotoxin boosts burst firing and catecholamine release in mouse chromaffin cells.

KEY POINTS: Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast-inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca(2+)-dependent secretion of catecholamines. Here, we report MCCs expressing large densities of neuronal fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no subthreshold pacemaker currents and can be slowly inactivated by 50% upon slight membrane depolarization. Reducing Nav1.3/Nav1.7 availability by tetrodotoxin or by sustained depolarization near rest leads to a switch from tonic to burst-firing patterns that give rise to elevated Ca(2+)-influx and increased catecholamine release. Spontaneous burst firing is also evident in a small percentage of control MCCs. Our results establish that burst firing comprises an intrinsic firing mode of MCCs that boosts their output. This occurs particularly when Nav channel availability is reduced by sustained splanchnic nerve stimulation or prolonged cell depolarizations induced by acidosis, hyperkalaemia and increased muscarine levels. ABSTRACT: Action potential (AP) firing in mouse chromaffin cells (MCCs) is mainly sustained by Cav1.3 L-type channels that drive BK and SK currents and regulate the pacemaking cycle. As secretory units, CCs optimally recruit Ca(2+) channels when stimulated, a process potentially dependent on the modulation of the AP waveform. Our previous work has shown that a critical determinant of AP shape is voltage-gated sodium channel (Nav) channel availability. Here, we studied the contribution of Nav channels to firing patterns and AP shapes at rest (-50 mV) and upon stimulation (-40 mV). Using quantitative RT-PCR and immunoblotting, we show that MCCs mainly express tetrodotoxin (TTX)-sensitive, fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no Na(+) current during slow ramp depolarizations. Time constants and the percentage of recovery from fast inactivation and slow entry into closed-state inactivation are similar to that of brain Nav1.3 and Nav1.7 channels. The fraction of available Nav channels is reduced by half after 10 mV depolarization from -50 to -40 mV. This leads to low amplitude spikes and a reduction in repolarizing K(+) currents inverting the net current from outward to inward during the after-hyperpolarization. When Nav channel availability is reduced by up to 20% of total, either by TTX block or steady depolarization, a switch from tonic to burst firing is observed. The spontaneous occurrence of high frequency bursts is rare under control conditions (14% of cells) but leads to major Ca(2+)-entry and increased catecholamine release. Thus, Nav1.3/Nav1.7 channel availability sets the AP shape, burst-firing initiation and regulates catecholamine secretion in MCCs. Nav channel inactivation becomes important during periods of high activity, mimicking stress responses.

Heterogeneous distribution of exocytotic microdomains in adrenal chromaffin cells resolved by high-density diamond ultra-microelectrode arrays.

Here we describe the ability of a high-density diamond microelectrode array targeted to resolve multi-site detection of fast exocytotic events from single cells. The array consists of nine boron-doped nanocrystalline diamond ultra-microelectrodes (9-Ch NCD-UMEA) radially distributed within a circular area of the dimensions of a single cell. The device can be operated in voltammetric or chronoamperometric configuration. Sensitivity to catecholamines, tested by dose-response calibrations, set the lowest detectable concentration of adrenaline to ∼5 µm. Catecholamine release from bovine or mouse chromaffin cells could be triggered by electrical stimulation or external KCl-enriched solutions. Spikes detected from the cell apex using carbon fibre microelectrodes showed an excellent correspondence with events measured at the bottom of the cell by the 9-Ch NCD-UMEA, confirming the ability of the array to resolve single quantal secretory events. Subcellular localization of exocytosis was provided by assigning each quantal event to one of the nine channels based on its location. The resulting mapping highlights the heterogeneous distribution of secretory activity in cell microdomains of 12-27 µm2. In bovine chromaffin cells, secretion was highly heterogeneous with zones of high and medium activity in 54% of the cell surface and zones of low or no activity in the remainder. The 'non-active' ('silent') zones covered 24% of the total and persisted for 6-8 min, indicating stable location. The 9-Ch NCD-UMEA therefore appears suitable for investigating the microdomain organization of neurosecretion with high spatial resolution.

Development and characterization of a diamond-insulated graphitic multi electrode array realized with ion beam lithography.

The detection of quantal exocytic events from neurons and neuroendocrine cells is a challenging task in neuroscience. One of the most promising platforms for the development of a new generation of biosensors is diamond, due to its biocompatibility, transparency and chemical inertness. Moreover, the electrical properties of diamond can be turned from a perfect insulator into a conductive material (resistivity ~mΩ·cm) by exploiting the metastable nature of this allotropic form of carbon. A 16­channels MEA (Multi Electrode Array) suitable for cell culture growing has been fabricated by means of ion implantation. A focused 1.2 MeV He+ beam was scanned on a IIa single-crystal diamond sample (4.5 × 4.5 × 0.5 mm3) to cause highly damaged sub-superficial structures that were defined with micrometric spatial resolution. After implantation, the sample was annealed. This process provides the conversion of the sub-superficial highly damaged regions to a graphitic phase embedded in a highly insulating diamond matrix. Thanks to a three-dimensional masking technique, the endpoints of the sub-superficial channels emerge in contact with the sample surface, therefore being available as sensing electrodes. Cyclic voltammetry and amperometry measurements of solutions with increasing concentrations of adrenaline were performed to characterize the biosensor sensitivity. The reported results demonstrate that this new type of biosensor is suitable for in vitro detection of catecholamine release.

Methodologies for Detecting Quantal Exocytosis in Adrenal Chromaffin Cells Through Diamond-Based MEAs.

Diamond-based multiarray sensors are suitable to detect in real-time exocytosis and action potentials from cultured, spontaneously firing chromaffin cells, primary hippocampal neurons, and midbrain dopaminergic neurons. Here, we focus on how amperometric measurements of catecholamine release are performed on micrographitic diamond multiarrays (µG-D-MEAs) with high temporal and spatial resolution by 16 electrodes simultaneously.

Multielectrode Arrays as a Means to Study Exocytosis in Human Platelets.

Platelets are probably the most accessible human cells to study exocytosis by amperometry. These cell fragments accumulate biological amines, serotonin in particular, using similar if not the same mechanisms as those employed by sympathetic, serotoninergic, and histaminergic neurons. Thus, platelets have been widely recognized as a model system to study certain neurological and psychiatric diseases. Platelets release serotonin by exocytosis, a process that entails the fusion of a secretory vesicle to the plasma membrane and that can be monitored directly by classic single cell amperometry using carbon fiber electrodes. However, this is a tedious technique because any given platelet releases only 4-8 secretory δ-granules. Here, we introduce and validate a diamond-based multielectrode array (MEA) device for the high-throughput study of exocytosis by human platelets. This is probably the first reported study of human tissue using an MEA, demonstrating that they are very interesting laboratory tools to assess alterations to exocytosis in neuropsychiatric diseases. Moreover, these devices constitute a valuable platform for the rapid testing of novel drugs that act on secretory pathways in human tissues.

Alpha-synuclein oligomers alter the spontaneous firing discharge of cultured midbrain neurons.

The aim of this work was to monitor the effects of extracellular α-synuclein on the firing activity of midbrain neurons dissociated from substantia nigra TH-GFP mice embryos and cultured on microelectrode arrays (MEA). We monitored the spontaneous firing discharge of the network for 21 days after plating and the role of glutamatergic and GABAergic inputs in regulating burst generation and network synchronism. Addition of GABA A , AMPA and NMDA antagonists did not suppress the spontaneous activity but allowed to identify three types of neurons that exhibited different modalities of firing and response to applied L-DOPA: high-rate (HR) neurons, low-rate pacemaking (LR-p), and low-rate non-pacemaking (LR-np) neurons. Most HR neurons were insensitive to L-DOPA, while the majority of LR-p neurons responded with a decrease of the firing discharge; less defined was the response of LR-np neurons. The effect of exogenous α-synuclein (α-syn) on the firing discharge of midbrain neurons was then studied by varying the exposure time (0-48 h) and the α-syn concentration (0.3-70 µM), while the formation of α-syn oligomers was monitored by means of AFM. Independently of the applied concentration, acute exposure to α-syn monomers did not exert any effect on the spontaneous firing rate of HR, LR-p, and LR-np neurons. On the contrary, after 48 h exposure, the firing activity was drastically altered at late developmental stages (14 days in vitro, DIV, neurons): α-syn oligomers progressively reduced the spontaneous firing discharge (IC50 = 1.03 µM), impaired burst generation and network synchronism, proportionally to the increased oligomer/monomer ratio. Different effects were found on early-stage developed neurons (9 DIV), whose firing discharge remained unaltered, regardless of the applied α-syn concentration and the exposure time. Our findings unravel, for the first time, the variable effects of exogenous α-syn at different stages of midbrain network development and provide new evidence for the early detection of neuronal function impairment associated to aggregated forms of α-syn.

Corrigendum: Alpha-synuclein oligomers alter the spontaneous firing discharge of cultured midbrain neurons.

[This corrects the article DOI: 10.3389/fncel.2023.1078550.].