Michael D. Boehler

NbActiv4 medium improvement to Neurobasal/B27 increases neuron synapse densities and network spike rates on multielectrode arrays

The most interesting property of neurons is their long-distance propagation of signals as spiking action potentials. Since 1993, Neurobasal/B27 has been used as a serum-free medium optimized for hippocampal neuron survival. Neurons on microelectrode arrays (MEA) were used as an assay system to increase spontaneous spike rates in media of different compositions. We find spike rates of 0.5 s(-1) (Hz) for rat embryonic hippocampal neurons cultured in Neurobasal/B27, lower than cultures in serum-based media and offering an opportunity for improvement. NbActiv4 was formulated by addition of creatine, cholesterol and estrogen to Neurobasal/B27 that synergistically produced an eightfold increase in spontaneous spike activity. The increased activity with NbActiv4 correlated with a twofold increase in immunoreactive synaptophysin bright puncta and GluR1 total puncta. Characteristic of synaptic scaling, immunoreactive GABAAbeta puncta also increased 1.5-fold and NMDA-R1 puncta increased 1.8-fold. Neuron survival in NbActiv4 equaled that in Neurobasal/B27, but with slightly higher astroglia. Resting respiratory demand was decreased and demand capacity was increased in NbActiv4, indicating less stress and higher efficiency. These results show that NbActiv4 is an improvement to Neurobasal/B27 for cultured networks with an increased density of synapses and transmitter receptors which produces higher spontaneous spike rates in neuron networks.

Added astroglia promote greater synapse density and higher activity in neuronal networks

Astroglia are known to potentiate individual synapses, but their contribution to networks is unclear. Here we examined the effect of adding either astroglia or media conditioned by astroglia on entire networks of rat hippocampal neurons cultured on microelectrode arrays. Added astroglia increased spontaneous spike rates nearly two-fold and glutamate-stimulated spiking by six-fold, with desensitization eliminated for bath addition of 25 microM glutamate. Astrocyte-conditioned medium partly mimicked the effects of added astroglia. Bursting behavior was largely unaffected by added astroglia except with added glutamate. Addition of the GABA(A) receptor antagonist bicuculline also increased spike rates but with more subtle differences between networks without or with added astroglia. This indicates that networks without added astroglia were inhibited greatly. In all conditions, the log-log distribution of spike rates fit well to linear distributions over three orders of magnitude. Networks with added astroglia shifted consistently toward higher spike rates. Immunostaining for GFAP revealed a linear increase with added astroglia, which also increased neuronal survival. The increased spike rates with added astroglia correlated with a 1.7-fold increase in immunoreactive synaptophysin puncta, and increases of six-fold for GABA(Abeta), two-fold for NMDA-R1 and two-fold for Glu-R1 puncta, with receptor clustering that indicated synaptic scaling. Together, these results indicate that added astroglia increase the density of synapses and receptors, and facilitate higher spike rates for many elements in the network. These effects are reproduced by glia-conditioned media, with the exception of glutamate-mediated transmission.

Neuron network activity scales exponentially with synapse density

Neuronal network output in the cortex as a function of synapse density during development has not been explicitly determined. Synaptic scaling in cortical brain networks seems to alter excitatory and inhibitory synaptic inputs to produce a representative rate of synaptic output. Here, we cultured rat hippocampal neurons over a three-week period to correlate synapse density with the increase in spontaneous spiking activity. We followed the network development as synapse formation and spike rate in two serum-free media optimized for either (a) neuron survival (Neurobasal/B27) or (b) spike rate (NbActiv4). We found that while synaptophysin synapse density increased linearly with development, spike rates increased exponentially in developing neuronal networks. Synaptic receptor components NR1, GluR1 and GABA-A also increase linearly but with more excitatory receptors than inhibitory. These results suggest that the brains information processing capability gains more from increasing connectivity of the processing units than increasing processing units, much as Internet information flow increases much faster than the linear number of nodes and connections.

Chronic electrical stimulation of cultured hippocampal networks increases spontaneous spike rates

We chronically stimulated hippocampal networks in culture for either 0, 1 or 3h/day between 7 and 22 days in culture in an effort to increase spontaneous spike rates and to give these networks some portion of external stimuli that brain networks receive during their formation. Chronic electrical stimulation of hippocampal networks on multi-electrode arrays (MEAs) increased spike rates 2-fold after 3 weeks of culture compared to cultures that received no external stimulation prior to recording. More than 90% of the spikes for all experimental conditions occurred within bursts. The frequency of spikes within a burst increased with time of stimulation during culture up to 2-fold higher (90Hz) compared to networks without chronic stimulation. However, spontaneous overall spike rates did not correlate well with the amount of stimulation either as h/day or proximity to the limited number of stimulation sites due to shorter burst duration with 3h/day stimulation. The results suggest that chronic stimulation applied during network development recruits activity at 50% more electrodes and enables higher rates of spontaneous activity within bursts in cultured hippocampal networks.

Hippocampal networks on reliable patterned substrates

Toward the goal of reproducible live neuronal networks, we investigated the influence of substrate patterns on neuron compliance and network activity. We optimized process parameters of micro-contact printing for reproducible geometric patterns of 10 μm wide lines of polylysine with 4, 6, or 8 connections at a constant square array of nodes overlying the recording electrodes of a multielectrode array (MEA). We hypothesized that an increase in node connections would give the network more inputs resulting in higher neuronal outputs as network spike rates. We also chronically stimulated these networks during development and added astroglia to enhance network activity. Our results show that despite frequent localization of neuron somata over the electrodes, the number of spontaneously active electrodes was reduced 3-fold compared to random networks, independent of pattern complexity. Of the electrodes active, the overall spike rate was independent of pattern complexity, consistent with homeostasis of activity. Lower mean burst rates were seen with higher levels of pattern complexity; however, burst durations increased 1.6-fold with pattern complexity (n=6027 bursts, p<0.001). Inter-burst interval and percentage of active electrodes displaying bursts also increased with pattern complexity. The extra-burst (non-burst or isolated) spike rate increased 4-fold with pattern complexity, but this relationship was reversed with either chronic stimulation or astroglia addition. These studies suggest for the first time that patterns which limit the distribution of branches and inputs are deleterious to activity in a hippocampal network, but that higher levels of pattern complexity promote non-burst activity and favor longer lasting, but fewer bursts.

Chronic network stimulation enhances evoked action potentials

Neurons cultured on multielectrode arrays almost always lack external stimulation except during the acute experimental phase. We have investigated the effects of chronic stimulation during the course of development in cultured hippocampal neural networks by applying paired pulses at half of the electrodes for 0, 1 or 3 r/day for 8 days. Spike latencies increased from 4 to 16 ms as the distance from the stimulus increased from 200 to 1700 microm, suggesting an average of four synapses over this distance. Compared to no chronic stimulation, our results indicate that chronic stimulation increased evoked spike counts per stimulus by 50% at recording sites near the stimulating electrode and increased the instantaneous firing rate. On trials where both pulses elicited responses, spike count was 40-80% higher than when only one of the pulses elicited a response. In attempts to identify spike amplitude plasticity, we found mainly amplitude variation with different latencies suggesting recordings from neurons with different identities. These data suggest plastic network changes induced by chronic stimulation that enhance the reliability of information transmission and the efficiency of multisynaptic network communication.

Toward a self-wired active reconstruction of the hippocampal trisynaptic loop: DG-CA3

The mammalian hippocampus functions to encode and retrieve memories by transiently changing synaptic strengths, yet encoding in individual subregions for transmission between regions remains poorly understood. Toward the goal of better understanding the coding in the trisynaptic pathway from the dentate gyrus (DG) to the CA3 and CA1, we report a novel microfabricated device that divides a micro-electrode array into two compartments of separate hippocampal network subregions connected by axons that grow through 3 × 10 × 400 μm tunnels. Gene expression by qPCR demonstrated selective enrichment of separate DG, CA3, and CA1 subregions. Reconnection of DG to CA3 altered burst dynamics associated with marked enrichment of GAD67 in DG and GFAP in CA3. Surprisingly, DG axon spike propagation was preferentially unidirectional to the CA3 region at 0.5 m/s with little reverse transmission. Therefore, select hippocampal subregions intrinsically self-wire in anatomically appropriate patterns and maintain their distinct subregion phenotype without external inputs.

The Mini Report: a Practical Tool to Address Lung Cancer Disparities in Rural Communities.

Community-based participatory research (CBPR) is an effective way to address cancer disparities in medically underserved populations. Our research demonstrates how CBPR principles were used to develop lung cancer and risk factor mini reports for a network of community coalitions in the Illinois Delta Region, a predominately rural region with high lung cancer disparities in southern Illinois. An academic-community partnership, including a community-based medical school, state public health department, and a healthcare system, used CBPR principles to translate epidemiological, behavioral, and demographic data into understandable, comprehensive, yet concise mini reports for each coalition. A cyclical and iterative process was used to draft, revise, and optimize these mini reports to raise awareness about lung cancer disparities in the community and to provide information to help guide the development of interventions that address these disparities. The use of CBPR principles was a successful way to create mini reports about local lung cancer disparities and risk factors that were usable in individual communities. Local coalitions used the mini reports to educate community members at local meetings, to guide strategic planning, and to disseminate information through their respective websites. Additionally, the process of creating these reports built trust among academic-community partners and provided additional avenues of engagement, such as the involvement of an academic partner in the strategic planning process of a local coalition. Using CBPR processes is an effective way to translate epidemiological data into a community-friendly format to address cancer disparities.

Abstract A40: The “Mini-Report”: Use of CBPR to create a practical tool to address lung cancer disparities in rural communities

Community Based Participatory Research (CBPR) principles were used to build an academic-community partnership to address lung cancer disparities in the Healthy Southern Illinois Delta Network (HSIDN), which includes 7 community coalitions in the southernmost 16 counties in Illinois. These counties are rural and medically underserved, and compared to IL as a whole, experience notably higher rates of smoking (27.1% vs. 16.9%), and lung cancer incidence (87.8 vs. 72.6/100,000) and mortality (66.8 vs. 54.4/100,000). By involving community members in all aspects of the research process and data dissemination, health disparity factors are more effectively identified and addressed. Our goal was to translate epidemiological data into community-specific lung cancer and behavioral risk factor “mini-reports” which could be used at the local level for education, resource allocation, and targeted intervention development. Methods: The partnership included an academic medical institution, a governmental agency, two community partners, and the HSIDN. Three work groups formed as part of the partnership infrastructure and two were vital in the development of these mini reports. The data work group (DWG) included members from the academic and governmental agency partners and was responsible for creating the reports. The CBPR work group included academic and community partners and was invaluable in providing feedback on report content, structure and intended audience suitability. Initial draft reports were based upon input from a partnership summit and DWG discussions. Draft report feedback tools were distributed to all coalition leaders in the HSIDN to assess content, display, motivation to action, and cultural appropriateness. The partnership9s steering committee also provided feedback to refine the mini-reports for use at the community level. Results: The DWG finalized two mini reports, both of which compared coalition-specific data to IL as a whole. The first report described the local cancer burden and included cancer incidence, mortality, survival by stage, population demographics, and lung cancer risk factors (e.g. smoking rates). The second report characterized the at-risk populations (e.g. veterans, those below poverty level) within the coalitions and included smoking rates by gender, youth smoking rates, and data on the effect of statewide smoke free laws on smoking rates and attitudes. Both reports used a mix of narrative text, bullet points, graphs, tables, and graphics. Feedback from the HSIDN coalition leaders indicated the reports effectively described the cancer burden and the content, relevancy, and usefulness of the reports was appropriate. HSIDN leaders noted a wide range of future uses for these reports. Discussion: These mini-reports translated epidemiological data for use at the community level for educational and other purposes. HSIDN use the reports for development of strategic planning, input in state-required community health plans, implementation of smoke-free initiatives, and grant writing. The creation process can serve as a template for other academic-community partnerships aiming to address cancer disparities using CBPR principles. The partnership successfully developed a series of coalition level reports to describe the local lung cancer burden and characterize risk factors to help coalitions create interventions to address disparities. The process of report creation, refinement and local dissemination was successful due to engagement of all partners (academic, government and community) and utilization of their expertise to ensure the reports could be used effectively in their communities. This in turn contributes to partnership sustainability by establishing trust for future efforts. Citation Format: Georgia Mueller, Whitney Zahnd, Kyle Garner, Ruth Heitkamp, Wiley Jenkins, Michael Boehler, Diane Land, David Steward. The “Mini-Report”: Use of CBPR to create a practical tool to address lung cancer disparities in rural communities. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr A40. doi:10.1158/1538-7755.DISP13-A40