Quantal Release of Dopamine and Action Potential Firing Detected in Midbrain Neurons by Multifunctional Diamond-Based Microarrays

Abstract

Micro-Graphitic Single Crystal Diamond Multi Electrode Arrays (μG-SCD-MEAs) have so far been used as amperometric sensors to detect catecholamines from chromaffin cells and adrenal gland slices. Besides having time resolution and sensitivity that are comparable with carbon fiber electrodes, that represent the gold standard for amperometry, μG-SCD-MEAs also have the advantages of simultaneous multisite detection, high biocompatibility and implementation of amperometric/potentiometric protocols, aimed at monitoring exocytotic events and neuronal excitability. In order to adapt diamond technology to record neuronal activity, the μG-SCD-MEAs in this work have been interfaced with cultured midbrain neurons to detect electrical activity as well as quantal release of dopamine (DA). μG-SCD-MEAs are based on graphitic sensing electrodes that are embedded into the diamond matrix and are fabricated using MeV ion beam lithography. Two geometries have been adopted, with 4 × 4 and 8 × 8 microelectrodes (20 μm × 3.5 μm exposed area, 200 μm spacing). In the amperometric configuration, the 4 × 4 μG-SCD-MEAs resolved quantal exocytosis from midbrain dopaminergic neurons. KCl-stimulated DA release occurred as amperometric spikes of 15 pA amplitude and 0.5 ms half-width, at a mean frequency of 0.4 Hz. When used as potentiometric multiarrays, the 8 × 8 μG-SCD-MEAs detected the spontaneous firing activity of midbrain neurons. Extracellularly recorded action potentials (APs) had mean amplitude of ∼-50 μV and occurred at a mean firing frequency of 0.7 Hz in 67% of neurons, while the remaining fired at 6.8 Hz. Comparable findings were observed using conventional MEAs (0.9 and 6.4 Hz, respectively). To test the reliability of potentiometric recordings with μG-SCD-MEAs, the D 2 -autoreceptor modulation of firing was investigated by applying levodopa ( L -DOPA, 20 μM), and comparing μG-SCD-MEAs, conventional MEAs and current-clamp recordings. In all cases, L -DOPA reduced the spontaneous spiking activity in most neurons by 70%, while the D 2 -antagonist sulpiride reversed this effect. Cell firing inhibition was generally associated with increased APs amplitude. A minority of neurons was either insensitive to, or potentiated by L -DOPA, suggesting that AP recordings originate from different midbrain neuronal subpopulations and reveal different modulatory pathways. Our data demonstrate, for the first time, that μG-SCD-MEAs are multi-functional biosensors suitable to resolve real-time DA release and AP firing in in vitro neuronal networks.

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

“…The carrier is equipped with a perfusion chamber containing the growing medium essential for the cell plating. The device is finally interfaced with the front-end electronic whose performances and characteristics are determined by the kind of measurement performed (amperometry or potentiometry) but are in general similar to those of commercial systems like the amplifier for carbon fibre electrodes or titanium-nitride (TiN) MEAs [32,79,81,109,115].…”

“…The micrographitized diamond MEA (16 channels), when used as amperometric sensors, have been employed to detect quantal release events not only from neuroendocrine chromaffin and PC12 cells [83], whose oxidation of granule content produces current events of hundreds of picoamperes and lasting tens of milliseconds, but also from cultured midbrain dopaminergic neurons: in this case, spike duration is completed within hundreds of microseconds [109]. Monitoring quantal exocytosis from dopaminergic neurons requires to increase the sampling frequency from 4 to > 25 kHz [109]. Spike half-time values have been estimated around 650 μs, in good agreement with those reported from CFEs [103].…”

“…Cultured rat hippocampal neurons, GT1-7 cells and chick ciliary ganglia neurons preserve their excitability when cultured on hydrogen-and oxygen-terminated diamond surfaces [9,84], providing that the diamond probes are coated with adhesive molecules (poly-D-lysine, poly-DL-ornithine, laminin) to favour cell anchoring. Feasibility of recordings from in vitro and in vivo preparations have been shown using BDD-MEA and 3D-nanostructured BDD-MEA [49,84], ultra-nanocrystalline nitrogen-doped diamond (NDD) MEA [116] and micrographitized diamond MEAs [109]. In the latter case, besides monitoring the spontaneous activity of midbrain neurons, we could provide a detailed analysis of the regulation of their firing properties by L-DOPA and the involvement of D 2 autoreceptors.…”

A biocompatible technique for magnetic field sensing at (sub)cellular scale using Nitrogen-Vacancy centers

“…Magnetometry in biological systems is of the utmost importance for fundamental biological science and medicine [1]. Mapping brain activity by recording magnetic fields produced by the electrical currents which are naturally occurring in the brain is of extreme interest [2,3], with direct applications in the timely detection and cure of psychic and neurodegenerative disorders [4][5][6]. Measuring the magnetic fields produced by electrical currents in the heart is also of the utmost importance [7], since this could lead to a new generation of non-invasive diagnostic and therapeutic techniques [8].…”

“…Magnetometers based on Nitrogen-Vacancy (NV) centers in diamond represent a valid alternative to SQUID-based magnetometry. Firstly, diamond offers the substantial advan-tage of being fully biocompatible [6,[11][12][13][14][15]. On the other side, NV centers are characterized by a peculiar electronic level structure that allows the optical detection of their microwave-driven spin resonances with a technique referred as Optically Detected Magnetic Resonance(ODMR) [16][17][18][19].…”