Christopher G. Langhammer

Automated Sholl analysis of digitized neuronal morphology at multiple scales: Whole cell Sholl analysis versus Sholl analysis of arbor subregions

The morphology of dendrites and the axon determines how a neuron processes and transmits information. Neurite morphology is frequently analyzed by Sholl analysis or by counting the total number of neurites and branch tips. However, the time and resources required to perform such analysis by hand is prohibitive for the processing of large data sets and introduces problems with data auditing and reproducibility. Furthermore, analyses performed by hand or using course‐grained morphometric data extraction tools can obscure subtle differences in data sets because they do not store the data in a form that facilitates the application of multiple analytical tools. To address these shortcomings, we have developed a program (titled “Bonfire”) to facilitate digitization of neurite morphology and subsequent Sholl analysis. Our program builds upon other available open‐source morphological analysis tools by performing Sholl analysis on subregions of the neuritic arbor, enabling the detection of local level changes in dendrite and axon branching behavior. To validate this new tool, we applied Bonfire analysis to images of hippocampal neurons treated with 25 ng/ml brain‐derived neurotrophic factor (BDNF) and untreated control neurons. Consistent with prior findings, conventional Sholl analysis revealed that global exposure to BDNF increases the number of neuritic intersections proximal to the soma. Bonfire analysis additionally uncovers that BDNF treatment affects both root processes and terminal processes with no effect on intermediate neurites. Taken together, our data suggest that global exposure of hippocampal neurons to BDNF results in a reorganization of neuritic segments within their arbors, but not necessarily a change in their number or length. These findings were only made possible by the neurite‐specific Sholl data returned by Bonfire analysis.

Neuroprotective Drug Riluzole Amplifies the Heat Shock Factor 1 (HSF1)- and Glutamate Transporter 1 (GLT1)-dependent Cytoprotective Mechanisms for Neuronal Survival

Heat shock factor 1 (HSF1) mediates the cellular response to stress to increase the production of heat shock protein (HSP) chaperones for proper protein folding, trafficking, and degradation; failure of this homeostatic mechanism likely contributes to neurodegeneration. We show that the neuroprotective drug riluzole increased the amount of HSF1 in NG108-15 neuroprogenitor cells by slowing the specific turnover of HSF1 and supporting a more robust and sustained activation of HSF1. Using Hsp70-luciferase as a functional readout of the activity of HSF1, we show that riluzole amplified the heat shock induction of the reporter gene with an optimal increase at 1 μm. Immunocytochemical staining and Western blot quantitation of HSP70 in NG108-15 neuroprogenitor cells and embryonic spinal cord neurons provided corroborative evidence that riluzole amplified the HSF1-dependent regulation of HSP70 expression. Parallel studies on the GLT1 glutamate transporter showed that riluzole increased GLT1-reporter and GLT1 protein expression and that the increase was enhanced by heat shock and coincident with the increased expression of HSP70 and HSP90. This result is consistent with the anti-glutamatergic profile of riluzole and the presence of multiple heat shock elements on the GLT1 gene promoter, suggesting that riluzole may modulate GLT1 expression through HSF1. The increased HSP chaperones and GLT1 transporter blunted glutamate-induced and N-methyl d-aspartate receptor-mediated excitotoxic death. In summary, we show that riluzole increased the amount and activity of HSF1 to boost the expression of HSPs and GLT1 for neuroprotection under stress.

Effects of substrate stiffness and cell density on primary hippocampal cultures.

Previous studies have shown that dendrites are influenced by substrate stiffness when neurons are plated in either pure or mixed cultures. However, because substrate rigidity can also affect other aspects of culture development known to impact dendrite branching, such as overall cell number, it is unclear whether substrate stiffness exerts a direct or indirect effect on dendrite morphology. In this study, we determine whether substrate stiffness plays a critical role in regulating dendrite branching independent of cell number. We plated primary mixed hippocampal cultures on soft and stiff gels, with Youngs moduli of 1 kPa and 7 kPa, respectively. We found that neurons plated on stiffer substrates showed increased branching relative to neurons grown on softer substrates at the same cell number. On the stiff gels, we also observed a cell number-dependent effect, in which increasing initial plating density decreased dendrite branching. This change correlates with an increase in extracellular glutamate. We concluded that both cell number and substrate stiffness play roles in determining dendrite branching, and that the two effects are independent of one another.

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 Alzheimers 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.

Regulation of dendrite arborization by substrate stiffness is mediated by glutamate receptors.

Brain injury or disease can initiate changes in local or global stiffness of brain tissue. While stiffness of the extracellular environment is known to affect the morphology and function of many cell types, little is known about how the dendrites of neurons respond to changes in brain stiffness. To assess how extracellular stiffness affects dendrite morphology, we took biomaterial and biomedical engineering approaches. We cultured mixed and pure hippocampal neurons on hydrogels composed of polyacrylamide (PA) of varying stiffnesses to mimic the effects of extracellular matrix stiffness on dendrite morphology. The majority of investigations of cortical and spinal cord neurons on soft hydrogels examined branching at early time points (days in vitro (DIV) 2–7), an important distinction from our study, where we include later time points that encompass the peak of branching (DIV 10–12). At DIV 12, dendrite branching was altered by stiffness for both pure and mixed neuronal cultures. Furthermore, we treated hippocampal cultures with glutamate receptor antagonists and with astrocyte-conditioned media. Blocking AMPA and NMDA receptors affected the changes in dendrite branching seen at varying rigidities. Moreover, extracellular factors secreted by astrocytes also change dendrite branching seen at varying rigidities. Thus, astrocytes and ionotropic glutamate receptors contribute to mechanosensing.

Medical student research exposure via a series of modular research programs

Background The falling percentage of doctors of medicine applying for National Institute of Health-funded research grants is 1 indicator that physician-scientists are a disappearing breed. This is occurring at a time when increased translational, disease-oriented, patient-oriented, and clinical research are national goals. One of the keys to providing sufficient numbers of physician-scientists to support this goal is the active targeting of medical students. We hypothesize that an improved research program infrastructure and responsiveness to changing student needs will increase student participation in research-oriented electives. Methods We have developed a student research program consisting of 2 Students Interested in Research noncredit electives (lecture and laboratory based), summer fellowships, support for year-out fellowships, and a Distinction in Research program that spans undergraduate medical education. Student participation and short-term research outcomes from fall 2004 through spring 2008 are analyzed to examine program efficacy. Results Students involved in the early parts of the program initially experienced higher application and success rates for summer funding opportunities, but as the program has matured, these rates have fallen in line with the class average. Independently, students participating in later portions of the program increasingly submit or publish a first author paper and have taken a year off for research during medical school. Overlap of participation in the programs is generally smaller than expected. Conclusion Although structured programs can provide step-wise research experiences of increasing intensity, students may not experience a training pipeline in which each stage relies on those before and after, and instead may sample an a la carte selection of research-based enrichment opportunities.

Assessing effects on dendritic arborization using novel Sholl analyses

Determining the shape of cell-specific dendritic arbors is a tightly regulated process that occurs during development. When this regulation is aberrant, which occurs during disease or injury, alterations in dendritic shape result in changes to neural circuitry. There has been significant progress on characterizing extracellular and intrinsic factors that regulate dendrite number by our laboratory and others. Generally, changes to the dendritic arbor are assessed by Sholl analysis or simple dendrite counting. However, we have found that this general method often overlooks local changes to the arbor. Previously, we developed a program (titled Bonfire) to facilitate digitization of neurite morphology and subsequent Sholl analysis and to assess changes to root, intermediate, and terminal neurites. Here, we apply these different Sholl analyses, and a novel Sholl analysis, to uncover previously unknown changes to the dendritic arbor when we overexpress an important regulator of dendrite branching, cytosolic PSD-95 interactor (cypin), at two developmental time points. Our results suggest that standard Sholl analysis and simple dendrite counting are not sufficient for uncovering local changes to the dendritic arbor.

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.

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.

Identification and Quantification of Skeletal Myotube Contraction and Association In Vitro by Video Microscopy

Skeletal muscle is the largest tissue in the body by weight and plays many roles in maintaining homeostasis and health. Ex vivo cell‐based experimental systems used to study muscle cell contraction, and others based on incorporation of cells into sensitive force transducers or electrophysiology equipment, are time‐consuming, invasive, and not universally available, slowing the pace of research. Video microscopy provides a noninvasive way to record the contractile behavior of skeletal muscle cells in vitro. We have developed a numerical procedure using image processing and pattern recognition algorithms, that makes it possible to quantify contractile behavior of multiple myotubes simultaneously, based on video data. We examined the ability of the program to identify movement using a simplified graphical model of myotube contraction and found that the programs success is dependent on the morphology and movement characteristics of the objects. However, the program performs optimally over the types of motions approximating those observed in culture and identifies contracting myotubes in sample videomicrographs of muscle cells in vitro. This program quantifies contractility on a population level, can be adapted for use in laboratories capable of digital video capture from a microscope, and may be coupled with other experimental techniques to supplement existing research tools.