Daniel A. Wagenaar

An extremely rich repertoire of bursting patterns during the development of cortical cultures

BackgroundWe have collected a comprehensive set of multi-unit data on dissociated cortical cultures. Previous studies of the development of the electrical activity of dissociated cultures of cortical neurons each focused on limited aspects of its dynamics, and were often based on small numbers of observed cultures. We followed 58 cultures of different densities – 3000 to 50,000 neurons on areas of 30 to 75 mm2 – growing on multi-electrode arrays (MEAs) during the first five weeks of their development.ResultsPlating density had a profound effect on development. While the aggregate spike detection rate scaled linearly with density, as expected from the number of cells in proximity to electrodes, dense cultures started to exhibit bursting behavior earlier in development than sparser cultures. Analysis of responses to electrical stimulation suggests that axonal outgrowth likewise occurred faster in dense cultures. After two weeks, the network activity was dominated by population bursts in most cultures. In contrast to previous reports, development continued with changing burst patterns throughout the observation period. Burst patterns were extremely varied, with inter-burst intervals between 1 and 300 s, different amounts of temporal clustering of bursts, and different firing rate profiles during bursts. During certain stages of development bursts were organized into tight clusters with highly conserved internal structure.ConclusionDissociated cultures of cortical cells exhibited a much richer repertoire of activity patterns than previously reported. Except for the very sparsest cultures, all cultures exhibited globally synchronized bursts, but bursting patterns changed over the course of development, and varied considerably between preparations. This emphasizes the importance of using multiple preparations – not just multiple cultures from one preparation – in any study involving neuronal cultures.These results are based on 963 half-hour-long recordings. To encourage further investigation of the rich range of behaviors exhibited by cortical cells in vitro, we are making the data available to other researchers, together with Matlab code to facilitate access.

Controlling Bursting in Cortical Cultures with Closed-Loop Multi-Electrode Stimulation

One of the major modes of activity of high-density cultures of dissociated neurons is globally synchronized bursting. Unlike in vivo, neuronal ensembles in culture maintain activity patterns dominated by global bursts for the lifetime of the culture (up to 2 years). We hypothesize that persistence of bursting is caused by a lack of input from other brain areas. To study this hypothesis, we grew small but dense monolayer cultures of cortical neurons and glia from rat embryos on multi-electrode arrays and used electrical stimulation to substitute for afferents. We quantified the burstiness of the firing of the cultures in spontaneous activity and during several stimulation protocols. Although slow stimulation through individual electrodes increased burstiness as a result of burst entrainment, rapid stimulation reduced burstiness. Distributing stimuli across several electrodes, as well as continuously fine-tuning stimulus strength with closed-loop feedback, greatly enhanced burst control. We conclude that externally applied electrical stimulation can substitute for natural inputs to cortical neuronal ensembles in transforming burst-dominated activity to dispersed spiking, more reminiscent of the awake cortex in vivo. This nonpharmacological method of controlling bursts will be a critical tool for exploring the information processing capacities of neuronal ensembles in vitro and has potential applications for the treatment of epilepsy.

Effective parameters for stimulation of dissociated cultures using multi-electrode arrays.

Electrical stimulation through multi-electrode arrays is used to evoke activity in dissociated cultures of cortical neurons. We study the efficacies of a variety of pulse shapes under voltage control as well as current control, and determine useful parameter ranges that optimize efficacy while preventing damage through electrochemistry. For any pulse shape, stimulation is found to be mediated by negative currents. We find that positive-then-negative biphasic voltage-controlled pulses are more effective than any of the other pulse shapes tested, when compared at the same peak voltage. These results suggest that voltage-control, with its inherent control over limiting electrochemistry, may be advantageous in a wide variety of stimulation scenarios, possibly extending to in-vivo experiments.

The Neurally Controlled Animat: Biological Brains Acting with Simulated Bodies

The brain is perhaps the most advanced and robust computation system known. We are creating a method to study how information is processed and encoded in living cultured neuronal networks by interfacing them to a computer-generated animal, the Neurally-Controlled Animat, within a virtual world. Cortical neurons from rats are dissociated and cultured on a surface containing a grid of electrodes (multi-electrode arrays, or MEAs) capable of both recording and stimulating neural activity. Distributed patterns of neural activity are used to control the behavior of the Animat in a simulated environment. The computer acts as its sensory system providing electrical feedback to the network about the Animats movement within its environment. Changes in the Animats behavior due to interaction with its surroundings are studied in concert with the biological processes (e.g., neural plasticity) that produced those changes, to understand how information is processed and encoded within a living neural network. Thus, we have created a hybrid real-time processing engine and control system that consists of living, electronic, and simulated components. Eventually this approach may be applied to controlling robotic devices, or lead to better real-time silicon-based information processing and control algorithms that are fault tolerant and can repair themselves.

Real-time multi-channel stimulus artifact suppression by local curve fitting

We describe an algorithm for suppression of stimulation artifacts in extracellular micro-electrode array (MEA) recordings. A model of the artifact based on locally fitted cubic polynomials is subtracted from the recording, yielding a flat baseline amenable to spike detection by voltage thresholding. The algorithm, SALPA, reduces the period after stimulation during which action potentials cannot be detected by an order of magnitude, to less than 2 ms. Our implementation is fast enough to process 60-channel data sampled at 25 kHz in real-time on an inexpensive desktop PC. It performs well on a wide range of artifact shapes without re-tuning any parameters, because it accounts for amplifier saturation explicitly and uses a statistic to verify successful artifact suppression immediately after the amplifiers become operational. We demonstrate the algorithms effectiveness on recordings from dense monolayer cultures of cortical neurons obtained from rat embryos. SALPA opens up a previously inaccessible window for studying transient neural oscillations and precisely timed dynamics in short-latency responses to electric stimulation.

Photochemical control of endogenous ion channels and cellular excitability

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

Precisely timed spatiotemporal patterns of neural activity in dissociated cortical cultures

Recurring patterns of neural activity, a potential substrate of both information transfer and transformation in cortical networks, have been observed in the intact brain and in brain slices. Do these patterns require the inherent cortical microcircuitry of such preparations or are they a general property of self-organizing neuronal networks? In networks of dissociated cortical neurons from rats--which lack evidence of the intact brains intrinsic cortical architecture--we have observed a robust set of spontaneously repeating spatiotemporal patterns of neural activity, using a template-matching algorithm that has been successful both in vivo and in brain slices. The observed patterns in cultured monolayer networks are stable over minutes of extracellular recording, occur throughout the cultures development, and are temporally precise within milliseconds. The identification of these patterns in dissociated cultures opens a powerful methodological avenue for the study of such patterns, and their persistence despite the topological and morphological rearrangements of cellular dissociation is further evidence that precisely timed patterns are a universal emergent feature of self-organizing neuronal networks.

MeaBench: A toolset for multi-electrode data acquisition and on-line analysis

We present a software suite, MeaBench, for data acquisition and online analysis of multi-electrode recordings, especially from micro-electrode arrays. Besides controlling data acquisition hardware, MeaBench includes algorithms for real-time stimulation artifact suppression and spike detection, as well as programs for online display of voltage traces from 60 electrodes and continuously updated spike raster plots. MeaBench features real-time output streaming, allowing easy integration with stimulator systems. We have been able to generate stimulation sequences in response to live neuronal activity with less than 20 ms lag time. MeaBench is open-source software, and is available for free public download at http://www.its.caltech.edu/~pinelab/wagenaar/meabench.html

Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4* Nicotinic Receptors

A leucine to alanine substitution (L9′A) was introduced in the M2 region of the mouse α4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, α4(L9′A)β2 nAChRs were ≥30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9′A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on α4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of α4or β2 subunits. Thalamic cultures and synaptosomes from L9′A mice were hypersensitive to nicotine-induced ion flux. L9′A mice were ∼15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9′A mice differed qualitatively from those in WT: L9′A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9′A mice were partial, whereas WT seizures were generalized. When L9′A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure ∼20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9′A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive α4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.

A versatile all-channel stimulator for electrode arrays, with real-time control

Over the last few decades, technology to record through ever increasing numbers of electrodes has become available to electrophysiologists. For the study of distributed neural processing, however, the ability to stimulate through equal numbers of electrodes, and thus to attain bidirectional communication, is of paramount importance. Here, we present a stimulation system for multi-electrode arrays which interfaces with existing commercial recording hardware, and allows stimulation through any electrode in the array, with rapid switching between channels. The system is controlled through real-time Linux, making it extremely flexible: stimulation sequences can be constructed on-the-fly, and arbitrary stimulus waveforms can be used if desired. A key feature of this design is that it can be readily and inexpensively reproduced in other labs, since it interfaces to standard PC parallel ports and uses only off-the-shelf components. Moreover, adaptation for use with in vivo multi-electrode probes would be straightforward. In combination with our freely available data-acquisition software, MeaBench, this system can provide feedback stimulation in response to recorded action potentials within 15 ms.