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 D2-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 D2-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.
Highlights
Dopamine (DA) plays fundamental roles in a variety of neurophysiological functions and neurological diseases
We have provided the first evidence that μG-SCD-multielectrode arrays (MEAs) can detect the quantal exocytosis of neuronal synaptic vesicles as well as spontaneous neuronal firing activity
The fact that the defects created by irradiation with MeV ions follow a typical distribution, which is characterized by the so-called “Bragg peak” and the ion end of range (Figure 1A), means the position of the graphitic electrodes can be modulated along the substrate depth, guaranteeing intrinsic electrical passivation due to the presence of the diamond cap layer
Summary
Dopamine (DA) plays fundamental roles in a variety of neurophysiological functions and neurological diseases. Since the early investigation of synaptic dysfunction is a target when attempting to understand the molecular mechanisms that lead to neurodegenerative processes, the development of mutifunctional sensing tools for simultaneous monitoring neurotransmitter release and electrical activity are extremely relevant for addressing key aspects of neurotransmission in the early stages of neurodegenerative diseases (Suzuki et al, 2013; Schirinzi et al, 2016; Castagnola et al, 2018; Ghiglieri et al, 2018; Picconi et al, 2018) In this regard, conventional multielectrode arrays (MEAs) have been employed to investigate the firing properties in SN pars compacta slices (Berretta et al, 2010), while amperometric detection of DA release from cultured neurons, was initially performed using CFEs (Pothos et al, 1998; Pothos, 2002; Staal et al, 2004; Mosharov and Sulzer, 2005). We have provided the first evidence that μG-SCD-MEAs can detect the quantal exocytosis of neuronal synaptic vesicles as well as spontaneous neuronal firing activity
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