Formation of Essential Ultrastructural Interface between Cultured Hippocampal Cells and Gold Mushroom-Shaped MEA- Toward ?IN-CELL? Recordings from Vertebrate Neurons

Abstract

Using cultured Aplysia neurons we recently reported on the development of a novel approach in which an extracellular, non-invasive multi-electrode-array system provides multisite, attenuated, intracellular recordings of subthreshold synaptic potentials, and action potentials (APs), the so called “IN-CELL” recording configuration (to differentiate it from intracellular recordings). Because of its non-invasive nature, the configuration can be used for long term semi intracellular electrophysiological monitoring of APs and synaptic potentials. Three principals converge to generate the IN-CELL configuration: (a) engulfment of approximately 1 μm size gold mushroom-shaped microelectrodes (gMμE) by the neurons, (b) formation of high seal resistance between the cell’s plasma membrane and the engulfed gMμE, and (c), autonomous localized increased conductance of the membrane patch facing the gMμE. Using dissociated rat hippocampal cultures we report here that the necessary morphological and ultrastructural relationships to generate the IN-CELL recording configuration are formed between hippocampal cells and the gMμEs. Interestingly, even <1 μm thin branches expand and engulf the gMμE structures. Recordings of spontaneous electrical activity revealed fast ∼2 ms, 0.04–0.75 mV positive monophasic APs (FPMP). We propose that the FPMP are attenuated APs generated by neurons that engulf gMμEs. Computer simulations of analog electrical circuits depicting the cell–gMμE configuration point out the parameters that should be altered to improve the neuron–gMμE electrical coupling.

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

“…was in the range of 100 μV, in the present study the average amplitudes for positive monophasic FPs were 1279 ± 146 μV (n = 148); for negative monophasic FPs 711 ± 66 μV (n = 45) and for the biphasic FPs 1221 ± 1045 μV (n = 557). The improved recording quality of the gMμE-MEA over substrate integrated planar MEA can be attributed to the increased seal resistance formed between the myotubes’ plasma membrane and the gMμE by its engulfment293031324348. Consistent with this hypothesis, the variability in the FP amplitudes is related to the level of gMμE engulfment by the cells43.…”

“…3). The FPs could be classified as biphasic, negative, or positive dominated potentials in a similar manner to those recorded by extracellular electrodes from non isopotential neurons or cardiomyocytes344748. Based on the “simple” shape of the FPs, their amplitude, and the fact that the dimensions and surface area of the gMμE are very small (~10 μm 2 ) with respect to the dimensions of the myotubes, it is reasonable to assume that most individual gMμE recorded FPs from a single myotube (Fig.…”

Multisite electrophysiological recordings by self-assembled loose-patch-like junctions between cultured hippocampal neurons and mushroom-shaped microelectrodes

“…In these studies2324252627282930 we demonstrated that cultured Aplysia neurons tightly engulf the gMμEs to form a high seal resistance. This, together with the increased conductance of the membrane patch that faces the gMμE, makes it possible to record action potentials and subthreshold synaptic potentials with qualities and biophysics similar to those obtained by perforated patch recording electrodes3132.…”

“…This high density electrode design was implemented with a correspondingly small 146 × 146 μm culturing surface area. As a result, the cultured rat hippocampal neurons did not develop typical network behavior and only sparse neuronal activity was monitored from one or two cells at a time28.…”

QSpike tools: a generic framework for parallel batch preprocessing of extracellular neuronal signals recorded by substrate microelectrode arrays

“…In addition, recent advances in real-time computing and in micro- and nanotechnologies opened brand new possibilities (Arsiero et al, 2007; Mazzatenta et al, 2007; Jain and Muthuswamy, 2008; Chen et al, 2009; Kim et al, 2009; Fendyur et al, 2011; Hai and Spira, 2012; Tian and Lieber, 2013). …”