Neuroelectronic interfacing with cultured multielectrode arrays toward a cultured probe

Efficient and selective electrical stimulation and recording of neural activity in peripheral, spinal, or central pathways requires multielectrode arrays at micrometer scale. ¿Cultured probe¿ devices are being developed, i.e., cell-cultured planar multielectrode arrays (MEAs). They may enhance efficiency and selectivity because neural cells have been grown over and around each electrode site as electrode-specific local networks. If, after implantation, collateral sprouts branch from a motor fiber (ventral horn area) and if they can be guided and contacted to each ¿host¿ network, a very selective and efficient interface will result. Four basic aspects of the design and development of a cultured probe, coated with rat cortical or dorsal root ganglion neurons, are described. First, the importance of optimization of the cell-electrode contact is presented. It turns out that impedance spectroscopy, and detailed modeling of the electrode-cell interface, is a very helpful technique, which shows whether a cell is covering an electrode and how strong the sealing is. Second, the dielectrophoretic trapping method directs cells efficiently to desired spots on the substrate, and cells remain viable after the treatment. The number of cells trapped is dependent on the electric field parameters and the occurrence of a secondary force, a fluid flow (as a result of field-induced heating). It was found that the viability of trapped cortical cells was not influenced by the electric field. Third, cells must adhere to the surface of the substrate and form networks, which are locally confined, to one electrode site. For that, chemical modification of the substrate and electrode areas with various coatings, such as polyethyleneimine (PEI) and fluorocarbon monolayers promotes or inhibits adhesion of cells. Finally, it is shown how PEI patterning, by a stamping technique, successfully guides outgrowth of collaterals from a neonatal rat lumbar spinal cord explant, after six days in cultur

Feed-Forward Propagation of Temporal and Rate Information between Cortical Populations during Coherent Activation in Engineered In Vitro Networks.

Transient propagation of information across neuronal assembles is thought to underlie many cognitive processes. However, the nature of the neural code that is embedded within these transmissions remains uncertain. Much of our understanding of how information is transmitted among these assemblies has been derived from computational models. While these models have been instrumental in understanding these processes they often make simplifying assumptions about the biophysical properties of neurons that may influence the nature and properties expressed. To address this issue we created an in vitro analog of a feed-forward network composed of two small populations (also referred to as assemblies or layers) of living dissociated rat cortical neurons. The populations were separated by, and communicated through, a microelectromechanical systems (MEMS) device containing a strip of microscale tunnels. Delayed culturing of one population in the first layer followed by the second a few days later induced the unidirectional growth of axons through the microtunnels resulting in a primarily feed-forward communication between these two small neural populations. In this study we systematically manipulated the number of tunnels that connected each layer and hence, the number of axons providing communication between those populations. We then assess the effect of reducing the number of tunnels has upon the properties of between-layer communication capacity and fidelity of neural transmission among spike trains transmitted across and within layers. We show evidence based on Victor-Purpura's and van Rossum's spike train similarity metrics supporting the presence of both rate and temporal information embedded within these transmissions whose fidelity increased during communication both between and within layers when the number of tunnels are increased. We also provide evidence reinforcing the role of synchronized activity upon transmission fidelity during the spontaneous synchronized network burst events that propagated between layers and highlight the potential applications of these MEMs devices as a tool for further investigation of structure and functional dynamics among neural populations

Information processing in dissociated neuronal cultures of rat hippocampal neurons

One of the major aims of Systems Neuroscience is to understand how the nervous system transforms sensory inputs into appropriate motor reactions. In very simple cases sensory neurons are immediately coupled to motoneurons and the entire transformation becomes a simple reflex, in which a noxious signal is immediately transformed into an escape reaction. However, in the most complex behaviours, the nervous system seems to analyse in detail the sensory inputs and is performing some kind of information processing (IP). IP takes place at many different levels of the nervous system: from the peripheral nervous system, where sensory stimuli are detected and converted into electrical pulses, to the central nervous system, where features of sensory stimuli are extracted, perception takes place and actions and motions are coordinated. Moreover, understanding the basic computational properties of the nervous system, besides being at the core of Neuroscience, also arouses great interest even in the field of Neuroengineering and in the field of Computer Science. In fact, being able to decode the neural activity can lead to the development of a new generation of neuroprosthetic devices aimed, for example, at restoring motor functions in severely paralysed patients (Chapin, 2004). On the other side, the development of Artificial Neural Networks (ANNs) (Marr, 1982; Rumelhart & McClelland, 1988; Herz et al., 1981; Hopfield, 1982; Minsky & Papert, 1988) has already proved that the study of biological neural networks may lead to the development and to the design of new computing algorithms and devices. All nervous systems are based on the same elements, the neurons, which are computing devices which, compared to silicon components, are much slower and much less reliable. How are nervous systems of all living species able to survive being based on slow and poorly reliable components? This obvious and na\uefve question is equivalent to characterizing IP in a more quantitative way. In order to study IP and to capture the basic computational properties of the nervous system, two major questions seem to arise. Firstly, which is the fundamental unit of information processing: 2 single neurons or neuronal ensembles? Secondly, how is information encoded in the neuronal firing? These questions - in my view - summarize the problem of the neural code. The subject of my PhD research was to study information processing in dissociated neuronal cultures of rat hippocampal neurons. These cultures, with random connections, provide a more general view of neuronal networks and assemblies, not depending on the circuitry of a neuronal network in vivo, and allow a more detailed and careful experimental investigation. In order to record the activity of a large ensemble of neurons, these neurons were cultured on multielectrode arrays (MEAs) and multi-site stimulation was used to activate different neurons and pathways of the network. In this way, it was possible to vary the properties of the stimulus applied under a controlled extracellular environment. Given this experimental system, my investigation had two major approaches. On one side, I focused my studies on the problem of the neural code, where I studied in particular information processing at the single neuron level and at an ensemble level, investigating also putative neural coding mechanisms. On the other side, I tried to explore the possibility of using biological neurons as computing elements in a task commonly solved by conventional silicon devices: image processing and pattern recognition. The results reported in the first two chapters of my thesis have been published in two separate articles. The third chapter of my thesis represents an article in preparation

Contribution of GABAergic Interneurons to the Development of Spontaneous Activity Patterns in Cultured Neocortical Networks

Periodic synchronized events are a hallmark feature of developing neuronal networks and are assumed to be crucial for the maturation of the neuronal circuitry. In the developing neocortex, the early network oscillations coincide with an excitatory action of the neurotransmitter gamma-aminobutyric acid (GABA). A relationship between the emerging inhibitory action of GABA and the gradual disappearance of early synchronized network activity has been previously suggested. Therefore we investigate the interplay between the action of GABA and spontaneous activity in cultured networks of the lateral or dorsal embryonic rat neocortex, which show considerable difference in the content of GABAergic neurons. Here we present the results of long-term monitoring of spontaneous electrical activity of cultured networks growing on microelectrode arrays and the time course of changes in GABA action using calcium imaging. All cultures studied displayed stereotyped synchronized burst events at the end of the first week in vitro. As the GABAA depolarizing action decreases, naturally or after bumetanide treatment, network activity in lateral cortex cultures changed from stereotypic bursting to more clustered and asynchronous activity patterns. Dorsal cortex cultures and cultures lacking GABAA-receptor mediated synaptic transmission, retained an immature synchronous firing pattern, but developed prominent intraburst oscillations (∼3–10 Hz). Large, mostly parvalbumin positive, GABAergic neurons dominate the GABAergic population in lateral cortex cultures. These large interneurons were virtually absent in dorsal cortex cultures. Based on these results, we suggest that the richly interconnected large GABAergic neurons contribute to desynchronize and temporally differentiate the spontaneous activity of cultured cortical networks

Extracellular stimulation window explained by a geometry-based model of the neuron-electrode contact

Extracellular stimulation of single cultured neurons which are completely sealing a microelectrode is usually performed using anodic or biphasic currents of at least 200 nA. However, recently obtained experimental data demonstrate the possibility to stimulate a neuron using cathodic current pulses with less amplitude. Also, a stimulation window is observed. These findings can be explained by a finite-element model which permits geometry-based electrical representation of the neuron-electrode interface and can be used to explore the required conditions for extracellular stimulation in detail. Modulation of the voltage sensitive channels in the sealing part of the membrane appears to be the key to successful cathodic stimulation. Furthermore, the upper limit of the stimulation window can be explained as a normal consequence of the neuronal membrane electrophysiology

Developing an In Vitro Assay for Detection and Characterization of Functional Connectivity within Transplantation Candidate Embryonic Stem Cell-Derived V2a Interneuron Networks

Facilitating plasticity after spinal cord injury tends to be the focus of most modern interventions for this condition. In particular, stem cell therapies attempt to both modulate and mimic some of the native plasticity after injury through multiple mechanisms. One such mechanism, the creation of new exogenous relay circuits bridging the injury, has been explored extensively, revealing serious impediments to its optimization and adoption for clinical settings. Our collaborator, the Sakiyama-Elbert group, has spent years addressing the first limitation, the variability of cellular graft composition, by perfecting protocols to generate embryonic stem cell (ESC)-derived populations of neurons with pre-determined genetic identity. Recently, they developed a protocol to develop highly-enriched populations of Chx10-expressing V2a interneurons (INs), a ventral interneuron population that has garnered recent interest due to its role in central pattern generating function and favorable phenotypic properties. This predominantly glutamatergic and long, ipsilaterally projecting population appears to be a prime candidate for transplantation therapies for SCI, especially for the creation of relay circuits that can potentially circumnavigate injuries. The research documented in this thesis attempts to begin to address the second limitation of stem cell transplantation therapy, our minimal understanding of intra-graft network connectivity after transplantation. Due to the limitations of current techniques for evaluating the connectivity of populations like ESC-derived V2a INs, the relationship between functional recovery and the functional properties of the novel circuits formed within the graft still eludes researchers. This thesis focuses on the development of an assay capable of rapidly detecting connectivity within ESC-derived candidate populations. By extending previous work in the stem cell field, we combine in vitro multi-electrode arrays (MEAs) with an extensively studied metric of functional connectivity, cross-correlation, to detect and characterize individual functional connections between ESC-derived neurons. We first validated this assay by culturing ESC-derived populations differentiated for increased expression of Chx10 on MEAs. We found that both dissociated and aggregated cultures formed functional busting networks with significant functional connectivity detected with the use of Between-Sample Analysis of Connectivity, a methodology originally developed for in vitro circadian networks. Aggregated networks, however, had much more consistent electrode coverage and individual neuron detection that dissociated networks. After this validation study, we characterized the functional connectivity within highly-enriched populations of ESC-V2a INs, comparing their connectivity to populations of ESC-MN/glia and mixed populations of ESC-V2a/MN/glia. We found that ESC-MN/glia aggregates formed active networks with a variety of activity and functional connectivity that was dependent on the transmission of glutamate. ESC-V2a INs could only survive out to the 4-week time point if they were grown in media conditioned with glial factors, but these cultures still lacked spontaneous extracellular activity. Mixed ESC-V2a/MN/glia populations formed the most active networks and had thousands of detectable connections which were also dependent on glutamate transmission. Application of glycine antagonist modulated network activity but the underlying cause is fairly inconclusive due to possible secondary effects. High growth factor concentrations in the growth media actually decreased network activity and detectable functional connections in the mixed populations. All of these findings in this proof of concept study collectively suggest that a mixture of ESC-V2a INs and ESC-MN/glia may be the most viable candidate for transplantation and sets the stage for future investigations into the manipulability of their connectivity with electrical stimulation, as well as scaled versions of this assay performed in combination with animal studies