Intra-burst firing characteristics as network state parameters

Introduction \ud In our group we are aiming to demonstrate learning and memory capabilities of cultured networks of cortical neurons. A first step is to identify parameters that accurately describe changes in the network due to learning. Usually, such parameters are calculated from the responses to test-stimuli before and after a learning experiment. We propose that parameters should be calculated from the spontaneous activity before and after a learning experiment, as the applying of test-stimuli itself may alter the network. Since bursting is dominant in our cultures, we have investigated its spatio-temporal structure. \ud \ud Methods \ud Networks of cortical neurons were cultured on a MEA. Over a period from 9 to 35 DIV, the spontaneous activity has been measured on a regular basis. Measurements on a single day are always continuous; otherwise cultures are kept in a stove under controlled conditions (37 ˚C, 5% CO2, 100% humidity). Network bursts were detected by analysing the Array-Wide Spiking Rate (AWSR, the sum of activity over all electrodes). Next, we estimated the instantaneous AWSR during a burst by convolving spike-occurrences with a Gaussian function. We investigated the changes in burst profiles over time by aligning them to their peak AWSR. In 4 hour recording sessions, we grouped the burst profiles over 1 hour, resulting in 4 average burst profiles per day. In addition, a sufficient amount of aligned bursts yielded enough data to calculate the contribution of each recording site. \ud \ud Results \ud The burst profiles, calculated over a period of 1 hour, generally show little variation (figure 1). In subsequent hours, the profiles gradually change shape. Over a period of days however, the shape can change dramatically (figure 2). The relatively slow changes over the period of hours indicate an underlying probabilistic structure in the AWSR during bursts. The apparent structure in the burst profiles result from the relationships between individual recording sites, and thus also on the connectivity in the neural network. This is revealed in more detail by showing the contributions of individual sites (figure 3). The spike envelopes have a shape that is too detailed to be described accurately by a small set of parameters. \ud \ud Discussion \ud The burst profiles prove to be stable over a period of one hour, and gradually change their shape over several hours, as has also been suggested in [1]. The day-to-day changes in burst profiles may be the result of these gradual changes, thereby suggesting an intrinsically changing network. However, they can also be the result of putting the cultures back in the stove. The spike envelopes per recording site offer more detailed descriptions of the network state than the burst profiles. This may however be the amount of detail required to reveal the changes made during learning experiments. A subsequent refinement can be made by identifying distinct subgroups of bursts, as has been suggested in [2]

Cultured cortical networks described by conditional firing probabilities

Networks of cortical neurons were grown over multi electrode arrays to enable simultaneous measu-rement of action potentials from 60 electrodes. All possible pairs of electrodes (i,j) were tested for syn-chronized activity. We calculated conditional firing probability (CFPi,j[τ]) as the probability of an action potential at electrode j at t=τ, given that a spike was detected at i at t=0. If a CFPi,j[τ] distribution clearly deviated from flat, electrodes i and j were considered related. A function was fitted to each CFP-curve to obtain parameters for strength and delay. In young cultures the set of identified relationships changed rather quickly. At 16 days in vitro (DIV) 50% of the set changed within one day. Beyond 25 DIV this set stabilized: during a period of a week more than 50% of the set remained intact. Most individual relationships developed rather gradually. Moreover, beyond 25 DIV relational strength appeared quite stable during periods of ≈ 10 hours, with coefficients of variation (100×SD/mean) of ≈ 25% on average. CFP analysis provides a robust method to describe the stable underlying probabilistic structure of highly varying spontaneous activity in cultured cortical networks. It may offer a suitable basis for plasticity studies, in which induced changes should exceed spontaneous fluctuations. CFP analysis is likely to describe the network in sufficient detail to detect subtle changes in individual relationships. Analysis of data continuously recorded for ≈ 6 weeks, showed that highest stability is reached after ≈ 25 DIV, suggesting the 4th and 5th week as a suitable period for plasticity studies.\ud \u

Patch-clamping mit Mikroöffnungen in Polyimide-Folien

Die Patchclamp-Technik ist die aussagekräftigste Methode zur Untersuchung der Funktion und Regulation von Ionenkanälen. Sie basiert auf der Bildung eines engen Kontaktes zwischen der Spitze einer Glasspipette und der Membran einer Zelle, an die die Pipette herangeführt wird. Aus dem engen Kontakt resultiert ein elektrischer Widerstand im Gigaohm-Bereich zwischen der Elektrolytlösung im Innern der Pipette und der die Zelle und Pipette umgebenden Elektrolytlösung. Trotz der weitverbreiteten Verwendung dieser Methode ist die wahre Natur dieses Kontaktes und der resultierenden hochohmigen „Seal“-Bildung noch immer nicht im Detail verstanden. Zudem ist die Patchclamp-Methode zeitaufwendig und erfordert erfahrene Anwender sowie gutausgerüstete Setups. Im Moment ist noch keine Vorrichtung beschrieben, die diesen “Cell-by-cell”-Assay vollautomatisch durchführt. Dies ist jedoch die Voraussetzung für Automatisierung, Miniaturisierung und Parallelisierung, um mit dieser Methode Hochdurchsatz-Untersuchungen von pharmazeutischen Substanzen durchführen zu können. Mehrere Gruppen berichten über einen Ansatz, der die Glasspipette durch eine mikromechanisch gefertigte Siliziumstruktur ersetzen soll. Sie verwenden ein dünnes Diaphragma, in das ein mikroskopisch kleines Loch (Durchmesser im Nano- und Mikrometer-Bereich) eingebracht ist. Darauf werden Lipidvesikel aufgebracht, die per Adhäsion die Umgebung der Mikroöffnung hochohmig abdichten. Mit dieser Methode lassen sich Einzelkanalströme messen

PEDOT–CNT Composite Microelectrodes for Recording and Electrostimulation Applications: Fabrication, Morphology, and Electrical Properties

Composites of carbon nanotubes and poly(3,4-ethylenedioxythiophene, PEDOT) and layers of PEDOT are deposited onto microelectrodes by electropolymerization of ethylenedioxythiophene in the presence of a suspension of carbon nanotubes and polystyrene sulfonate. Analysis by FIB and SEM demonstrates that CNT–PEDOT composites exhibit a porous morphology whereas PEDOT layers are more compact. Accordingly, capacitance and charge injection capacity of the composite material exceed those of pure PEDOT layers. In vitro cell culture experiments reveal excellent biocompatibility and adhesion of both PEDOT and PEDOT–CNT electrodes. Signals recorded from heart muscle cells demonstrate the high S/N ratio achievable with these electrodes. Long-term pulsing experiments confirm stability of charge injection capacity. In conclusion, a robust fabrication procedure for composite PEDOT–CNT electrodes is demonstrated and results show that these electrodes are well suited for stimulation and recording in cardiac and neurophysiological research

Extracellular electrical signals in a neuron-surface junction: model of heterogeneous membrane conductivity

Signals recorded from neurons with extracellular planar sensors have a wide range of waveforms and amplitudes. This variety is a result of different physical conditions affecting the ion currents through a cellular membrane. The transmembrane currents are often considered by macroscopic membrane models as essentially a homogeneous process. However, this assumption is doubtful, since ions move through ion channels, which are scattered within the membrane. Accounting for this fact, the present work proposes a theoretical model of heterogeneous membrane conductivity. The model is based on the hypothesis that both potential and charge are distributed inhomogeneously on the membrane surface, concentrated near channel pores, as the direct consequence of the inhomogeneous transmembrane current. A system of continuity equations having non-stationary and quasi-stationary forms expresses this fact mathematically. The present work performs mathematical analysis of the proposed equations, following by the synthesis of the equivalent electric element of a heterogeneous membrane current. This element is further used to construct a model of the cell-surface electric junction in a form of the equivalent electrical circuit. After that a study of how the heterogeneous membrane conductivity affects parameters of the extracellular electrical signal is performed. As the result it was found that variation of the passive characteristics of the cell-surface junction, conductivity of the cleft and the cleft height, could lead to different shapes of the extracellular signals

A Novel In Vitro Sensing Configuration for Retinal Physiology Analysis of a Sub-Retinal Prosthesis

This paper presents a novel sensing configuration for retinal physiology analysis, using two microelectrode arrays (MEAs). In order to investigate an optimized stimulation protocol for a sub-retinal prosthesis, retinal photoreceptor cells are stimulated, and the response of retinal ganglion cells is recorded in an in vitro environment. For photoreceptor cell stimulation, a polyimide-substrate MEA is developed, using the microelectromechanical systems (MEMS) technology. For ganglion cell response recording, a conventional glass-substrate MEA is utilized. This new sensing configuration is used to record the response of retinal ganglion cells with respect to three different stimulation methods (monopolar, bipolar, and dual-monopolar stimulation methods). Results show that the geometrical relation between the stimulation microelectrode locations and the response locations seems very low. The threshold charges of the bipolar stimulation and the monopolar stimulation are in the range of 10∼20 nC. The threshold charge of the dual-monopolar stimulation is not obvious. These results provide useful guidelines for developing a sub-retinal prosthesis

Spontaneous Oscillatory Rhythm in Retinal Activities of Two Retinal Degeneration (rd1 and rd10) Mice

Previously, we reported that besides retinal ganglion cell (RGC) spike, there is ~ 10 Hz oscillatory rhythmic activity in local field potential (LFP) in retinal degeneration model, rd1 mice. The more recently identified rd10 mice have a later onset and slower rate of photoreceptor degeneration than the rd1 mice, providing more therapeutic potential. In this study, before adapting rd10 mice as a new animal model for our electrical stimulation study, we investigated electrical characteristics of rd10 mice. From the raw waveform of recording using 8×8 microelectrode array (MEA) from in vitro-whole mount retina, RGC spikes and LFP were isolated by using different filter setting. Fourier transform was performed for detection of frequency of bursting RGC spikes and oscillatory field potential (OFP). In rd1 mice, ~10 Hz rhythmic burst of spontaneous RGC spikes is always phase-locked with the OFP and this phase-locking property is preserved regardless of postnatal ages. However, in rd10 mice, there is a strong phase-locking tendency between the spectral peak of bursting RGC spikes (~5 Hz) and the first peak of OFP (~5 Hz) across different age groups. But this phase-locking property is not robust as in rd1 retina, but maintains for a few seconds. Since rd1 and rd10 retina show phase-locking property at different frequency (~10 Hz vs. ~5 Hz), we expect different response patterns to electrical stimulus between rd1 and rd10 retina. Therefore, to extract optimal stimulation parameters in rd10 retina, first we might define selection criteria for responding rd10 ganglion cells to electrical stimulus