Mapping Micro-Circuits with Extra-Cellular Arrays in Three Layered Cortex
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1
Max-Planck-Institute for Brain Research, Department Laurent, Germany
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2
Max-Planck-Institute for Brain Research, Department Laurent, Germany
Motivation
Brain activity is generated and propagated via a large repertoire of transmembrane currents with different time scales. These currents shape the electric potential as measured, for example, by extra-cellular recordings. Despite the wealth of information embedded in these potentials and the abundant usage of extra-cellular probes, only limited information is usually extracted: high frequency spike shapes indicate the occurrence of action potentials in single neurons and local field potential report the summed synaptic activity of large populations of neurons. Here we focus on slow potentials, indicative of synaptic currents, and examine whether we can, from them, extract morphological properties of single neurons with multi-electrode array (MEAs).
We developed a preparation of three-layered cortex in turtle combined with MEAs. As in mammalian hippocampus and piriform cortex, excitatory neuron somata form a single layer – layer 2 – whose ventral boundary lies 50-100µm dorsal to the ependymal surface. This planar structure is ideally suited for electrode array recordings, providing potential access to tens of thousands of neurons over a square millimeter. Because the turtle brain tolerates anoxia relatively well, we can also use an intact brain preparation with eyes attached in a dish (Fig 1A). Such a preparation allows us to study cortical circuits under realistic sensory drive (Fig 1B), while benefiting from the high stability, accessibility and ease of manipulation of in vitro approaches.
Material and Methods
Turtles were anesthetized, decapitated and their heads transferred into cooled turtle Ringer solution. The skull was opened and the brain extracted with the eyes attached. The eyes were hemisected and visual stimulation was projected directly onto the retina. Cortex was flattened and placed on a MEA (pia facing up) with thalamic input fibers intact. Extracellular spikes were sorted into putative single units (Fig 1C). Individual neurons were simultaneously recorded using whole-cell patch-clamp techniques, to gain access to and control membrane potential and label the neurons.
Results
A typical recording yielded 300-800 sorted neurons. When spike-triggered averaging the raw extracellular voltage traces of single neurons, we observed that action potentials were usually followed by low frequency potentials (Fig 1D). We termed these spike induced fields (SIFs). SIFs had either positive or negative polarity with kinetics similar to those of synaptic currents (fast rise, slow decay). Interestingly, the SIF corresponding to each sorted spike had a unique spatio-temporal signature over the MEA. We verified that SIFs were generated by single neurons by patching individual neurons and controlling their spiking activity selectively. SIF polarity corresponded to inhibitory and excitatory neurons, identified from their intrinsic electrical and morphological properties. SIFs were post-synaptic in origin for blocking synaptic currents pharmacologically abolished SIFs while leaving spike waveforms intact. We next compared the reconstructed 3D morphology of patched neurons with the spatial distribution of the SIFs corresponding to their spikes and found a good correspondence. SIF amplitude was greater in locations in which axon collateral distribution was denser and smaller when axon collaterals were far from the recording MEA surface. By using volume conductance theory, we could estimate the SIF spatial distribution over the MEA from the reconstructed axonal morphology and compare it to that measured. Finally, we applied our method to examine the distribution of axonal projections in L3 of turtle dorsal cortex. By examining hundreds of simultaneously sampled neurons (Fig 1E), we found a strong bias towards neurons with axonal projections from the medial to the lateral zone of cortex. Such a bias could explain the consistent observation of medially propagating waves in this cortex.
Discussion
We show that information about single neuron identity (I vs. E) and morphology can be extracted from extra-cellular signals. Such information gathered from hundreds of neurons recorded simultaneously can be used to examine statistical aspects of micro-circuit structure in single experiments.
Figure legend
MEA recordings from intact turtle brains. (A) Top view of a turtle brain ex-vivo. (B) Cortical responses (raw traces of all channels shifted in y) to visual stimulation of a static image (gray arrow). (C) Spike template of one neuron and its corresponding SIF (D). Note the different time scales in C and D. (E) Spiking responses of ~380 sorted neurons to a series of static image presentations.
Acknowledgements
This work was supported by the Max Planck Society, the European Research Council (ERC) and the Minerva foundation.
Keywords:
Cortex,
propagating waves,
turtle,
axonal projections
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
oral
Topic:
MEA Meeting 2016
Citation:
Shein-Idelson
M,
Hemberger
M,
Pammer
L and
Laurent
G
(2016). Mapping Micro-Circuits with Extra-Cellular Arrays in Three Layered Cortex.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00031
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Mark Shein-Idelson, Max-Planck-Institute for Brain Research, Department Laurent, Frankfurt a.M., Germany, Mark.Shein-Idelson@brain.mpg.de