EXTRACELLULAR RECORDINGS OF SPIKES Information about spiking is typically extracted from the high frequency band (>300-500Hz) of extracellular potentials. Since these high-frequency signals generally stem from an unknown number of spiking neurons, it is called multi-unit activity (MUA). The low-frequency part (<300 Hz) of extracellular potentials is called the local field potential (LFP). High-pass filtering (at least at 300 Hz). (From Buzsaki et al., 2012)
The typical shape of an extracellular action potential (EAP) is: a sharp, deep dip (sodium phase) followed by a shallower, but longer-lasting, positive bump (potassium phase). As for the LFP, the EAP is seen to have an inverted sign apically compared to basally. (From Pettersen et al 2010)
Neuron-electrode interface: The cell membrane is represented with an equivalent model based on Hodgkin-Huxley model of the squid axon. Detailed characterizations of the electrode model for various materials have been carried out. Usually, in vivo models focus less on the electrode properties themselves, but more on the electric field generated by current sources in a conductive volume (for review see Obien et al., 2015). [The point-contact model, derived from Robinson (1968), Nelson et al. (2008), Hierlemann et al. (2011)]
In single-unit recordings sharp electrodes are positioned close to a neuronal soma. For such recordings the interpretation of the measurements is straightforward, but complications arise when more than one neuron contribute to the recorded extracellular potential. (From Quian Quiroga 2007)
It has to be considered: a) Intracellular and extracellular recordings from a neuron receiving both an excitatory and an inhibitory connection. b) Endogenous generation of APs c) APs train determined by excitatory drive d) Aps train determined by disinhibition e) Combination of excitation and inhibition without reaching the spike threshold. (From Spira and Hai, 2013)
Extracellular recordings provide little damage to the neuronal membranes and allow stable long-term recordings. Studying microcircuit processes requires simultaneously monitoring the activity of large numbers of individual neurons in multiple brain areas. While recording from every neuron in the brain is an unreasonable goal, recording from statistically representative samples of identified neurons from several local areas is feasible with currently available and emerging technologies. This is indeed a high-priority goal in systems neuroscience. Currently, electrode arrays can record from large numbers of neurons and monitor local neural circuits at work.
MEA (Multi-Electrode Array) Historical issues: -The first design of MEA recordings was introduced by Thomas et al., 1972 (2 x 15 array of 30 microelectrodes 7μm 2 in size and 100 μm apart). - in 1977 Gross and colleagues used another set of MEA (36 microelectrodes, 100 or 200 μm apart). - Pine et al. In 1980 correlated the intracellular and extracellular events by simultaneous recordings. - Only in the last two decades the application of the MEA technique spread, taking advantage of the commercial availability of MEA systems. - Thomas CA Jr, Springer PA, Loeb GE, Berwald-Netter Y, Okun LM. A miniature microelectrode array to monitor the bioelectric activity of cultured cells. Exp Cell Res 1972 -Pine J. Recording action potentials from cultured neurons with extracellular microcircuit electrodes. J Neurosci Methods 1980 - Liu MG, Chen XF, He T, Li Z, Chen J. Use of multi-electrode array recordings in studies of network synaptic plasticity in both time and space. Neurosci Bull 2012
Ideally, all materials used in MEA fabrication should have: 1) Good biocompatibility 2) Good electrical properties for high signal-to-noise ratio (SNR) 1) Good transparency for observation of organotypic or acute brain slices 2) Low cost (micro-fabrication technology is required: photolitography, metal deposition techniques...) Typically, electrodes are made with metallic conductors such as gold (Au), titanium nitride (TiN), platinum (Pt), stainless steel, aluminium (Al), and alloys like iridium oxide (IrOx). Since the electrodes are on the micrometer scale, it is a challenge to achieve low electrode impedance modifying the surface with porous conductive materials allows to drastically decrease electrode impedance and improve neuronal recordings (Obien et al., 2015).
In 1998 Egert et al. reported a novel planar MEA of 60 microelectrodes: the MEA60 is the primary product of the MEA system commercially provided by Multi Channel Systems (Reutlingen, Germany). In 1999 Oka et al. reported another new planar multi-electrode dish (MED) probe with 64 microelectrodes. (From Liu et al., 2012)
In order to reduce the distance between the recording electrodes and active cells (avoiding the dead cell layer ), Thiebaud et al. introduced a 60-channel MEA with 3-dimensional tipshaped protruding microelectrodes (3D-MEA). (From Liu et al., 2012)
MEA in vivo Severe connectivity limitation, as connections cannot be wired out on all four sides of the array (Obien et al., 20014). In single-unit recordings sharp electrodes are positioned close to a neuronal soma. For such recordings the interpretation of the measurements is straightforward, but complications arise when more than one neuron contribute to the recorded extracellular potential. The use of two (stereotrode), four (tetrode) or more (multishank) close-neighbored recording sites allows for improved spike sorting (triangulation). One can sort spike trains from tens of neurons from single tetrodes and hundreds of neurons with multishank electrodes. (Pettersen2010) Various geometrical arrangements: linear, matrix.
Tetrode Because most anatomical wiring is local, the majority of neuronal interactions, and thus computation, occur in a small volume (as the cortical columns). In addition, the use of two or more recording sites allows for the triangulation of distances. This can be achieved using four spaced wires (tetrodes, 50 μm spread). E.g.: tetrodes can derive hippocampal CA1 pyramidal neurons as far away as 140 um lateral to the cell body. A cylinder with a radius of 140 μm contains 1000 neurons in the rat cortex, which is the number of theoretically recordable cells by a single electrode. Yet, in practice, only a small fraction of the neurons can be reliably separated. Thus, there is a large gap between the numbers of routinely recorded and theoretically recorded neurons. (From Buzsaki, 2004)
Multi-shank probes E.g. High-density recording of unit activity in the somatosensory cortex of the rat, using an eight-shank silicone probe in layer 5. (From Buzsaki, 2004)
Linear probes Silicon linear probes in which 16-32 recording sites are equally spaced along the shank of the electrode thus simultaneously recording the neuronal activity across several layers within a brain region. (From Buzsaki et al., 2003) For example, this approach is useful in the neocortex, where the cortical modules are organized in columns, vertically.
Examples of linear probes: University of Michigan 16 channel silicon probe Acreo VSAMUEL 32 channel silicon probe 50 µm 750 μm 1550 μm (e.g. Biella et al., 2002a,b)
Microdrive systems e.g. The Echorn Matrix System microdrives (set up at the laboratory of Prof. Dr. R. Echorn at the Dept of Neurophysics Philipps-University Marburg, Germany) Separation of 80-100 μm
Our system: Echorn Matrix 16 electrodes (4x4) Separation of 100 μm Platinum-tungsten (95%-5%) Electrode outer diameter 80 μm Electrode tip 25 μm (From Laurens et al., 2010)
Chronic implants The increase in the number of the electrodes and sites led to an increase in the volume of the wire tether used to transmit the signal to an external amplifier. This would interfere with animal normal behavior. So the first step was to multiplex the signal from 32 independet recording channels into one wire channel (32:1 ratio). A more thorough solution consist in wireless transmission of up to 64 channels to a receiver placed 3-4 m away from the implanted animal. (From Szuts et al., 2011) A common problem is recording stability, since tissue damage can result in microglia and astrocytes encapsulation of the electrode (100-200μm) leading to deterioration of the signal.
Concluding remarks: Electrode size: Sizes of published microelectrodes range from 5 to 50 μm in diameter. Larger electrodes have a higher possibility of getting physically near the neurons and of picking up higher amplitude spikes. However, large electrodes (>50 μm) can average out a neuron s spatially localized peak signal amplitude with nearby smaller amplitude signals. This reduce peak signals, which can result in a lower SNR. Electrode size also affects the electrode impedance, which in turn determines electrode noise and SNR. (Obien et al., 2004)
MEA (Multi-Electrode Array) Advantages - Gathering large amounts of spatial information on the internal dynamics of networks with multisite recordings - Long-term analysis of the spatiotemporal distribution of network-level electrical activity - Multisite stimulation and recordings - Evaluation of network physiological properties (as spontaneous activity) and the pharmacological effects of compounds - Stable recording that is less sensitive to factors such as mechanical vibrations - Tremendous variety of research approach (acute brain slices, organotypic slice cultures, dissociated cell cultures, whole brain preparations and in vivo)
MEA (Multi-Electrode Array) and Disadvantages: - Only extracellular events (spikes and LFP) can be recorded - The signal-to-noise ratio is greatly affected by the quality of MEA probes (and far below that of intracellular or patch-clamp recording) - The problem of spatial resolution: the inter-electrode distance is usually to large to give a accurate estimation of events within microcircuits