Patch-clamp | EPFL Graph Search

vignette|Enregistrement (picoAmpère en fonction du temps) d'un patch-clamp montrant les passages entre deux états de conductance d'un canal ionique : fermé (ligne du haut) et ouvert (ligne du bas). Patch-clamp est un terme anglais désignant une technique électrophysiologique d'enregistrement des courants ioniques transitant à travers les membranes cellulaires. Cette technique consiste à mettre en continuité électrique une micro-pipette en verre (diamètre de contact de l'ordre de 1 µm) remplie d'une solution ionique de composition définie avec la membrane d'une cellule vivante isolée. Les cellules étudiées peuvent être des cellules excitables comme les neurones, les fibres musculaires et les cellules beta du pancréas, ou des cellules non excitables, qui présentent elles aussi à leur surface des canaux ioniques. En transférant la séquence génique (transfection) d'un canal ionique d'intérêt dans une cellule, la technique permet d'étudier le fonctionnement de tout canal ionique. Cette te

Synaptic and Cellular Organization of Layer 1 of the Developing Rat Somatosensory Cortex

Shruti Muralidhar

In this thesis, we systematically characterised the circuitry, cellular composition and synaptic connectivity of layer 1 (L1) of the somatosensory cortex of juvenile rats (post natal days 13-16). Electrophysiological and morphological parameters were measured using single and multi-cell patch-clamp electrophysiology followed by histochemical staining and 3D morphological reconstruction. Guided by the Petilla convention, we classified cells in term of their firing patterns, identifying five electrophysiological types: classical Non-Accommodating Cells (cNAC), classical Accommodating Cells (cAC), burst Non-Accommodating Cells (bNAC), classical Stuttering cells (cSTUT) and classical Irregular Spiking cells (cIR). We created first a subjective and then an objective classification of different morphological cell types. The subjective classification identified the following types – NGC-DA, NGC-SA, HAC, DAC, LAC and SAC. The initial classification was refined and validated using PCA and LDA. We also studied connectivity patterns among L1 cells. Our results showed that a majority of the cells were connected by a slow GABAB mediated inhibitory connection, with a fast GABAA component. Multiple repetitions of stimulation produced a prominent run-down of postsynaptic potential amplitudes, which was not seen in perforated patch-clamp recordings that maintained the intracellular milieu. We propose that the run-down was due to the dilution of intracellular components and depletion of secondary messengers necessary for signalling of postsynaptic metabotropic GABAB receptor mediated responses. 3D morphological reconstructions of these pairs of cells, showed the presence of multi-synapse connections with an average of 9 putative contacts per detected electrophysiological connection. The average coupling co-efficient of gap junctions was 0.054 ± 0.03 with symmetrical conductance. In additional studies, we investigated patterns of inter-laminar connectivity, the modulation of apical dendritic activity by L1 neurons and patterns of gene expression in L1. We hope that the results these studies – single cell electrophysiology, morphology and connectivity analysis - provide useful hints for future studies of neocortical L1.

EPFL2013

Light-Evoked Modulation of Neuronal Activity with Conjugated Polymers

Diego Ghezzi, Mattia Nova

Organic bioelectronics aims at the intimate integration between organic materials and biological tissues in order to obtain a targeted functional outcome. In the last decades, conductive polymers have been successfully employed to interface excitable cells through biocompatible probes and devices. We previously reported the possibility to culture primary neurons on photosensitive conjugated polymers, such as poly(3-hexylthiophene) (P3HT), without altering either the polymer or the neuronal properties. These neurons displayed a depolarizing response to a brief (< 50 ms) green light stimulus and a consequent control over the action potential firing rate. The same interface was also shown to elicit activation of retinal circuitry and ganglion cell firing in explanted degenerate retinas upon light stimulation. However, while neuronal excitation has been extensively proved via planar or protruding electrodes, carbon nanotubes, and conjugated polymers, the hyperpolarization of neurons and silencing of their firing activity has been achieved with optogenetics, but rarely documented for functionalized planar devices and structures. We show here a method to induce both depolarization and hyperpolarization of cells interfaced with conjugated polymers upon prolonged (500 ms) light stimulation. Patch-clamp experiments showed that HEK293 cells grown on P3HT displayed a biphasic response composed of an initial depolarization followed by a sustained hyperpolarization during light illumination. We also documented that the amplitude of the light-induced hyperpolarization was reduced in the absence of intracellular potassium, but was insensitive to changes in the intracellular chloride concentration. A prolonged illumination was equally able to hyperpolarize primary neurons cultured over P3HT and significantly reduced both spontaneous and electrically elicited action potential frequency. We further assessed the translatability of our findings to tissue stimulation by describing the modulation of ganglion cell firing in explanted blind retinas from Royal College of Surgeons rats, a model of Retinitis pigmentosa. By coupling conjugated polymers with multi-electrode arrays and conventional metal electrodes, single-unit activity of ganglion cells was inhibited by prolonged illumination. We similarly investigated the phenomenon in acute hippocampal brain slices placed onto a conjugated polymer thin film. These results indicate that conjugated polymers can be used to non-invasively excite and inhibit the electrical activity of cells cultured on its surface. Furthermore, the translation of our findings from in vitro cultures to different central nervous system tissues, suggests a potential application of these organic devices as a tool for modulation of neuronal activity in vivo.

2015