Antagonism of purinergic signalling improves recovery from traumatic brain injury.

The recent public awareness of the incidence and possible long-term consequences of traumatic brain injury only heightens the need to develop effective approaches for treating this neurological disease. In this report, we identify a new therapeutic target for traumatic brain injury by studying the role of astrocytes, rather than neurons, after neurotrauma. We use in vivo multiphoton imaging and show that mechanical forces during trauma trigger intercellular calcium waves throughout the astrocytes, and these waves are mediated by purinergic signalling. Subsequent in vitro screening shows that astrocyte signalling through the 'mechanical penumbra' affects the activity of neural circuits distant from the injury epicentre, and a reduction in the intercellular calcium waves within astrocytes restores neural activity after injury. In turn, the targeting of different purinergic receptor populations leads to a reduction in hippocampal cell death in mechanically injured organotypic slice cultures. Finally, the most promising therapeutic candidate from our in vitro screen (MRS 2179, a P2Y1 receptor antagonist) also improves histological and cognitive outcomes in a preclinical model of traumatic brain injury. This work shows the potential of studying astrocyte signalling after trauma to yield new and effective therapeutic targets for treating traumatic brain injury.

Altered uric acid levels and disease states.

Altered serum uric acid concentrations, both above and below normal levels, have been linked to a number of disease states. An abnormally high uric acid level has been correlated with gout, hypertension, cardiovascular disease, and renal disease, whereas a reduced uric acid concentration has been linked to multiple sclerosis, Parkinson's disease, Alzheimer's disease, and optic neuritis. Historically, uric acid has been considered a marker of these disease states. Recent studies, however, have provided evidence that uric acid may actually play a role in the development or progression of such diseases. As a result, the manipulation of uric acid concentrations is now either included in, or being investigated for, the treatment of a variety of disease states.

A topographically modified substrate-embedded MEA for directed myotube formation at electrode contact sites.

Myoblast fusion into functionally distinct myotubes, and their subsequent integration with the nervous system, is a poorly understood phenomenon with important applications in basic science research, skeletal muscle tissue engineering, and cell-based biosensor development. We have previously demonstrated the ability of microelectrode arrays (MEAs) to record the extracellular action potentials of myotubes, and we have shown that this information reveals the presence of multiple, electrophysiologically independent myotubes even in unstructured cultures where there is extensive physical contact between cells (Langhammer et al., Biotechnol Prog 27:891-895, 2011). In this paper, we explore the ability of microscale topographical trenches to guide the myoblast alignment and fusion processes and use our findings to create a substrate-embedded MEA containing topographical trenches that are able to direct myotube contractility to specific locations. By combining substrate-embedded MEA technology with topographical patterns, we have developed a lab-on-a-chip test bed for the non-invasive examination of myotubes.

Semiautomated analysis of dendrite morphology in cell culture.

Quantifying dendrite morphology is a method for determining the effect of biochemical pathways and extracellular agents on neuronal development and differentiation. Quantification can be performed using Sholl analysis, dendrite counting, and length quantification. These procedures can be performed on dendrite-forming cell lines or primary neurons grown in culture. In this protocol, we describe the use of a set of computer programs to assist in quantifying many aspects of dendrite morphology, including changes in total and localized arbor complexity.

Protection from glutamate-induced excitotoxicity by memantine.

This study investigates whether the uncompetitive N-methyl-D-aspartic acid receptor antagonist, memantine, is able to protect dissociated cortical neurons from glutamate-induced excitotoxicity (GIE). Treatment with glutamate resulted in a significant loss of synchronization of neuronal activity as well as a significant increase in the duration of synchronized bursting events (SBEs). By administering memantine at the same time as glutamate, we were able to completely prevent these changes to the neuronal activity. Pretreatment with memantine was somewhat effective in preventing changes to the culture synchronization but was unable to fully protect the synchronization of electrical activity between neurons that showed high levels of synchronization prior to injury. Additionally, memantine pretreatment was unable to prevent the increase in the duration of SBEs caused by GIE. Thus, the timing of memantine treatment is important for conferring neuroprotection against glutamate-induced neurotoxicity. Finally, we found that GIE leads to a significant increase in the burst duration. Our data suggest that this may be due to an alteration in the inhibitory function of the neurons.

Measurement of synchronous activity by microelectrode arrays uncovers differential effects of sublethal and lethal glutamate concentrations on cortical neurons.

We grew cultures of rat cortical cells on microelectrode arrays to investigate the effects of glutamate-mediated neurotoxicity as a model of traumatic brain injury. Treatment with two different concentrations of glutamate, 175 and 250 µM, led to different outcomes. Cultures treated with 250 µM glutamate suffered a loss in overall activity that was not seen in cultures treated with 175 µM glutamate. An analysis of the changes in the synchronization of action potential firing between electrodes, however, revealed a loss of synchronization in subsets of electrode pairs treated with both the higher and lower concentrations of glutamate. We found that this loss of action potential synchronization was dependent on the initial amount of synchronization prior to injury. Finally, our data suggest that the synchronization of electrical activity as well as the susceptibility to loss of firing synchrony is independent of the distance between neurons in a network.

Skeletal myotube integration with planar microelectrode arrays in vitro for spatially selective recording and stimulation: a comparison of neuronal and myotube extracellular action potentials.

Microelectrode array (MEA) technology holds tremendous potential in the fields of biodetection, lab-on-a-chip applications, and tissue engineering by facilitating noninvasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces.

Automated Sholl analysis of digitized neuronal morphology at multiple scales.

Neuronal morphology plays a significant role in determining how neurons function and communicate. Specifically, it affects the ability of neurons to receive inputs from other cells and contributes to the propagation of action potentials. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease, schizophrenia, and mental retardation. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction. Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations. The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.