Spindle oscillations in communicating axons within a reconstituted hippocampal formation are strongest in CA3 without thalamus.

Spindle-shaped waves of oscillations emerge in EEG scalp recordings during human and rodent non-REM sleep. The association of these 10-16 Hz oscillations with events during prior wakefulness suggests a role in memory consolidation. Human and rodent depth electrodes in the brain record strong spindles throughout the cortex and hippocampus, with possible origins in the thalamus. However, the source and targets of the spindle oscillations from the hippocampus are unclear. Here, we employed an in vitro reconstruction of four subregions of the hippocampal formation with separate microfluidic tunnels for single axon communication between subregions assembled on top of a microelectrode array. We recorded spontaneous 400-1000 ms long spindle waves at 10-16 Hz in single axons passing between subregions as well as from individual neurons in those subregions. Spindles were nested within slow waves. The highest amplitudes and most frequent occurrence suggest origins in CA3 neurons that send feed-forward axons into CA1 and feedback axons into DG. Spindles had 50-70% slower conduction velocities than spikes and were not phase-locked to spikes suggesting that spindle mechanisms are independent of action potentials. Therefore, consolidation of declarative-cognitive memories in the hippocampus may be separate from the more easily accessible consolidation of memories related to thalamic motor function.

A reversible early oxidized redox state that precedes macromolecular ROS damage in aging nontransgenic and 3xTg-AD mouse neurons.

The brain depends on redox electrons from nicotinamide adenine dinucleotide (reduced form; NADH) to produce ATP and oxyradicals (reactive oxygen species [ROS]). Because ROS damage and mitochondrial dysregulation are prominent in aging and Alzheimer's disease (AD) and their relationship to the redox state is unclear, we wanted to know whether an oxidative redox shift precedes these markers and leads to macromolecular damage in a mouse model of AD. We used the 3xTg-AD mouse model, which displays cognitive deficits beginning at 4 months. Hippocampal/cortical neurons were isolated across the age span and cultured in common nutrients to control for possible hormonal and vascular differences. We found an increase of NAD(P)H levels and redox state in nontransgenic (non-Tg) neurons until middle age, followed by a decline in old age. The 3xTg-AD neurons maintained much lower resting NAD(P)H and redox states after 4 months, but the NADH regenerating capacity continuously declined with age beginning at 2 months. These redox characteristics were partially reversible with nicotinamide, a biosynthetic precursor of NAD+. Nicotinamide also protected against glutamate excitotoxicity. Compared with non-Tg neurons, 3xTg-AD neurons had more mitochondria/neuron and lower glutathione (GSH) levels that preceded age-related increases in ROS levels. These GSH deficits were again reversible with nicotinamide in 3xTg-AD neurons. Surprisingly, low macromolecular ROS damage was only elevated after 4 months in the 3xTg-AD neurons if antioxidants were removed. The present data suggest that a more oxidized redox state and a lower antioxidant GSH defense can be dissociated from neuronal ROS damage, changes that precede the onset of cognitive deficits in the 3xTg-AD model.

The flow of axonal information among hippocampal sub-regions 2: patterned stimulation sharpens routing of information transmission.

The sub-regions of the hippocampal formation are essential for episodic learning and memory formation, yet the spike dynamics of each region contributing to this function are poorly understood, in part because of a lack of access to the inter-regional communicating axons. Here, we reconstructed hippocampal networks confined to four subcompartments in 2D cultures on a multi-electrode array that monitors individual communicating axons. In our novel device, somal, and axonal activity was measured simultaneously with the ability to ascertain the direction and speed of information transmission. Each sub-region and inter-regional axons had unique power-law spiking dynamics, indicating differences in computational functions, with abundant axonal feedback. After stimulation, spiking, and burst rates decreased in all sub-regions, spikes per burst generally decreased, intraburst spike rates increased, and burst duration decreased, which were specific for each sub-region. These changes in spiking dynamics post-stimulation were found to occupy a narrow range, consistent with the maintenance of the network at a critical state. Functional connections between the sub-region neurons and communicating axons in our device revealed homeostatic network routing strategies post-stimulation in which spontaneous feedback activity was selectively decreased and balanced by decreased feed-forward activity. Post-stimulation, the number of functional connections per array decreased, but the reliability of those connections increased. The networks maintained a balance in spiking and bursting dynamics in response to stimulation and sharpened network routing. These plastic characteristics of the network revealed the dynamic architecture of hippocampal computations in response to stimulation by selective routing on a spatiotemporal scale in single axons.

GTP energy dependence of endocytosis and autophagy in the aging brain and Alzheimer's disease.

Increased interest in the aging and Alzheimer's disease (AD)-related impairments in autophagy in the brain raise important questions about regulation and treatment. Since many steps in endocytosis and autophagy depend on GTPases, new measures of cellular GTP levels are needed to evaluate energy regulation in aging and AD. The recent development of ratiometric GTP sensors (GEVALS) and findings that GTP levels are not homogenous inside cells raise new issues of regulation of GTPases by the local availability of GTP. In this review, we highlight the metabolism of GTP in relation to the Rab GTPases involved in formation of early endosomes, late endosomes, and lysosomal transport to execute the autophagic degradation of damaged cargo. Specific GTPases control macroautophagy (mitophagy), microautophagy, and chaperone-mediated autophagy (CMA). By inference, local GTP levels would control autophagy, if not in excess. Additional levels of control are imposed by the redox state of the cell, including thioredoxin involvement. Throughout this review, we emphasize the age-related changes that could contribute to deficits in GTP and AD. We conclude with prospects for boosting GTP levels and reversing age-related oxidative redox shift to restore autophagy. Therefore, GTP levels could regulate the numerous GTPases involved in endocytosis, autophagy, and vesicular trafficking. In aging, metabolic adaptation to a sedentary lifestyle could impair mitochondrial function generating less GTP and redox energy for healthy management of amyloid and tau proteostasis, synaptic function, and inflammation.

Age-Related Oxidative Redox and Metabolic Changes Precede Intraneuronal Amyloid-ß Accumulation and Plaque Deposition in a Transgenic Alzheimer's Disease Mouse Model.

BACKGROUND: Many identified mechanisms could be upstream of the prominent amyloid-ß (Aß) plaques in Alzheimer's disease (AD). OBJECTIVE: To profile the progression of pathology in AD. METHODS: We monitored metabolic signaling, redox stress, intraneuronal amyloid-ß (iAß) accumulation, and extracellular plaque deposition in the brains of 3xTg-AD mice across the lifespan. RESULTS: Intracellular accumulation of aggregated Aß in the CA1 pyramidal cells at 9 months preceded extracellular plaques that first presented in the CA1 at 16 months of age. In biochemical assays, brain glutathione (GSH) declined with age in both 3xTg-AD and non-transgenic controls, but the decline was accelerated in 3xTg-AD brains from 2 to 4 months. The decline in GSH correlated exponentially with the rise in iAß. Integrated metabolic signaling as the ratio of phospho-Akt (pAkt) to total Akt (tAkt) in the PI3kinase and mTOR pathway declined at 6, 9, and 12 months, before rising at 16 and 20 months. These pAkt/tAkt ratios correlated with both iAß and GSH levels in a U-shaped relationship. Selective vulnerability of age-related AD-genotype-specific pAkt changes was greatest in the CA1 pyramidal cell layer. To demonstrate redox causation, iAß accumulation was lowered in cultured middle-age adult 3xTg-AD neurons by treatment of the oxidized redox state in the neurons with exogenous cysteine. CONCLUSION: The order of pathologic progression in the 3xTg-AD mouse was loss of GSH (oxidative redox shift) followed by a pAkt/tAkt metabolic shift in CA1, iAß accumulation in CA1, and extracellular Aß deposition. Upstream targets may prove strategically more effective for therapy before irreversible changes.

Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport.

Respiratory enzyme complex dysfunction is mechanistically involved in mitochondrial failure leading to neurodegenerative disease, but the pathway is unclear. Here, age-related differences in mitochondrial respiration were measured in both whole and permeabilized neurons from 9-month and 24-month adult rat cortex cultured in common conditions. After permeabilization, respiration increased in both ages of neurons with excess substrates. To dissect specific deficiencies in the respiratory chain, inhibitors for each respiratory chain complex were used to isolate their contributions. Relative to neurons from 9-month rats, in neurons isolated from 24-month rats, complexes I, III, and IV were more sensitive to selective inhibition. Flux control point analysis identified complex I in neurons isolated from 24-month rats as the most sensitive to endogenous substrate availability. The greatest age-related deficit in flux capacity occurred at complex IV with a 29% decrease in neurons isolated from 24-month rats relative to those from 9-month rats. The deficits in complexes I and III may contribute to a redox shift in the quinone pool within the electron transport chain, further extending these age-related deficits. Together these changes could lead to an age-related catastrophic decline in energy production and neuronal death.

The Flow of Axonal Information Among Hippocampal Subregions: 1. Feed-Forward and Feedback Network Spatial Dynamics Underpinning Emergent Information Processing.

The tri-synaptic pathway in the mammalian hippocampus enables cognitive learning and memory. Despite decades of reports on anatomy and physiology, the functional architecture of the hippocampal network remains poorly understood in terms of the dynamics of axonal information transfer between subregions. Information inputs largely flow from the entorhinal cortex (EC) to the dentate gyrus (DG), and then are processed further in the CA3 and CA1 before returning to the EC. Here, we reconstructed elements of the rat hippocampus in a novel device over an electrode array that allowed for monitoring the directionality of individual axons between the subregions. The direction of spike propagation was determined by the transmission delay of the axons recorded between two electrodes in microfluidic tunnels. The majority of axons from the EC to the DG operated in the feed-forward direction, with other regions developing unexpectedly large proportions of feedback axons to balance excitation. Spike timing in axons between each region followed single exponential log-log distributions over two orders of magnitude from 0.01 to 1 s, indicating that conventional descriptors of mean firing rates are misleading assumptions. Most of the spiking occurred in bursts that required two exponentials to fit the distribution of inter-burst intervals. This suggested the presence of up-states and down-states in every region, with the least up-states in the DG to CA3 feed-forward axons and the CA3 subregion. The peaks of the log-normal distributions of intra-burst spike rates were similar in axons between regions with modes around 95 Hz distributed over an order of magnitude. Burst durations were also log-normally distributed around a peak of 88 ms over two orders of magnitude. Despite the diversity of these spike distributions, spike rates from individual axons were often linearly correlated to subregions. These linear relationships enabled the generation of structural connectivity graphs, not possible previously without the directional flow of axonal information. The rich axonal spike dynamics between subregions of the hippocampus reveal both constraints and broad emergent dynamics of hippocampal architecture. Knowledge of this network architecture may enable more efficient computational artificial intelligence (AI) networks, neuromorphic hardware, and stimulation and decoding from cognitive implants.

Designing Neural Networks in Culture: Experiments are described for controlled growth, of nerve cells taken from rats, in predesigned geometrical patterns on laboratory culture dishes.

Technology has advanced to where it is possible to design and grow-with predefined geometry and surprisingly good fidelity-living networks of neurons in culture dishes. Here we overview the elements of design, emphasizing the lithographic techniques that alter the cell culture surface which in turn influences the attachment and growth of the neural networks. Advanced capability in this area makes it possible to design networks of desired complexity. Other issues addressed include the influence of glial cells and media on activity and the potential for extending the designs into three dimensions. Investigators are advancing the art and science of analyzing and controlling through stimulation the function of the neural networks, including the ability to take advantage of their geometric form in order to influence functional properties.

Age-Related Intraneuronal Aggregation of Amyloid-ß in Endosomes, Mitochondria, Autophagosomes, and Lysosomes.

This work provides new insight into the age-related basis of Alzheimer's disease (AD), the composition of intraneuronal amyloid (iAß), and the mechanism of an age-related increase in iAß in adult AD-model mouse neurons. A new end-specific antibody for Aß45 and another for aggregated forms of Aß provide new insight into the composition of iAß and the mechanism of accumulation in old adult neurons from the 3xTg-AD model mouse. iAß levels containing aggregates of Aß45 increased 30-50-fold in neurons from young to old age and were further stimulated upon glutamate treatment. iAß was 8 times more abundant in 3xTg-AD than non-transgenic neurons with imaged particle sizes following the same log-log distribution, suggesting a similar snow-ball mechanism of intracellular biogenesis. Pathologically misfolded and mislocalized Alz50 tau colocalized with iAß and rapidly increased following a brief metabolic stress with glutamate. AßPP-CTF, Aß45, and aggregated Aß colocalized most strongly with mitochondria and endosomes and less with lysosomes and autophagosomes. Differences in iAß by sex were minor. These results suggest that incomplete carboxyl-terminal trimming of long Aßs by gamma-secretase produced large intracellular deposits which limited completion of autophagy in aged neurons. Understanding the mechanism of age-related changes in iAß processing may lead to application of countermeasures to prolong dementia-free health span.

Global Metabolic Shifts in Age and Alzheimer's Disease Mouse Brains Pivot at NAD+/NADH Redox Sites.

Age and Alzheimer's disease (AD) share some common features such as cognitive impairments, memory loss, metabolic disturbances, bioenergetic deficits, and inflammation. Yet little is known on how systematic shifts in metabolic networks depend on age and AD. In this work, we investigated the global metabolomic alterations in non-transgenic (NTg) and triple-transgenic (3xTg-AD) mouse brain hippocampus as a function of age by using untargeted Ultrahigh Performance Liquid Chromatography-tandem Mass Spectroscopy (UPLC-MS/MS). We observed common metabolic patterns with aging in both NTg and 3xTg-AD brains involved in energy-generating pathways, fatty acids oxidation, glutamate, and sphingolipid metabolism. We found age-related downregulation of metabolites from reactions in glycolysis that consumed ATP and in the TCA cycle, especially at NAD+/NADH-dependent redox sites, where age- and AD-associated limitations in the free NADH may alter reactions. Conversely, metabolites increased in glycolytic reactions in which ATP is produced. With age, inputs to the TCA cycle were increased including fatty acid ß-oxidation and glutamine. Overall age- and AD-related changes were > 2-fold when comparing the declines of upstream metabolites of NAD+/NADH-dependent reactions to the increases of downstream metabolites (p = 10-5, n = 8 redox reactions). Inflammatory metabolites such as ceramides and sphingosine-1-phosphate also increased with age. Age-related decreases in glutamate, GABA, and sphingolipid were seen which worsened with AD genetic load in 3xTg-AD brains, possibly contributing to synaptic, learning- and memory-related deficits. The data support the novel hypothesis that age- and AD-associated metabolic shifts respond to NAD(P)+/NAD(P)H redox-dependent reactions, which may contribute to decreased energetic capacity.

Reversibility of Age-related Oxidized Free NADH Redox States in Alzheimer's Disease Neurons by Imposed External Cys/CySS Redox Shifts.

Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer's disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by 89% to levels corresponding to the young neurons. Using FLIM as a non-invasive approach, we were able to measure the reversibility of aging subcellular free NADH levels in live neurons. Our results suggest a potential reductive treatment to reverse the loss of free NADH in old and Alzheimer's neurons.

Age- and AD-related redox state of NADH in subcellular compartments by fluorescence lifetime imaging microscopy.

Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer's disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 to 41% more reducing NADH redox state than cytoplasm for each age, genotype, and sex. Aging diminished the mitochondrial free NADH concentration in NTg neurons by 43% and in 3xTg-AD by 50%. The lower free NADH with age suggests a decline in capacity to regenerate free NADH for energetic supply to power oxidative phosphorylation which further worsens in AD. Applying this non-invasive approach, we showed the most explicit measures yet of bioenergetic deficits in free NADH with aging at the subcellular level in live neurons from in-bred mice and an AD model.

Age-related changes to tumor necrosis factor receptors affect neuron survival in the presence of beta-amyloid.

Inflammation including local accumulations of tumor necrosis factor alpha (TNF-alpha) is a part of Alzheimer's disease pathology and may exacerbate age-related neurodegeneration. Most studies on TNF-alpha and TNF neuronal receptors are conducted by using embryonic neurons. Few studies consider age-related deficits that may occur in neurons. Age-related changes in susceptibility to TNF-alpha through TNF receptor 1 (TNFR1) and receptor 2 (TNFR2) expression could increase susceptibility to beta-amyloid (1-42, Abeta42). Evidence is conflicting about which receptor mediates survival and/or apoptosis. We determined how aging affects receptor expression in cultured adult rat cortical neurons. Old neurons were more susceptible to Abeta42 toxicity than middle-aged neurons, and the addition of TNF-alpha was neuroprotective in middle-aged neurons, but exacerbated the toxicity from Abeta42 in old neurons. These pathologic and protective responses in old and middle-aged neurons, respectively, correlated with higher starting TNFR1 and TNFR2 mRNA levels in old vs. middle-aged neurons. Middle-aged neurons treated with TNF-alpha plus Abeta42 did not show an increase in either TNFR1 or TNFR2 mRNA, but old neurons showed an up-regulation in TNFR2 mRNA and not TNFR1 mRNA. Despite these mRNA changes, surface immunoreactivity of both TNFR1 and TNFR2 increased with the dose of TNF-alpha in middle-aged neurons. However, middle-aged neurons treated with TNF-alpha plus Abeta42 showed an up-regulation in both TNFR1 and TNFR2 surface expression, whereas old neurons failed to up-regulate surface expression of either receptor. These findings support the hypothesis that age-related changes in TNF-alpha surface receptor expression contribute to the neuronal loss associated with inflammation in Alzheimer's disease.

Age-related decreases in NAD(P)H and glutathione cause redox declines before ATP loss during glutamate treatment of hippocampal neurons.

Age-related glutamate excitotoxicity depends in an unknown manner on active mitochondria, which are key determinants of the cellular redox potential. Compared with embryonic and middle-aged neurons, old-aged rat hippocampal neurons have a lower resting reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and a lower redox ratio (NAD(P)H/flavin adenine nucleotide). Glutamate treatment resulted in an initial increase in NAD(P)H concentrations in all ages, followed by a profound calcium-dependent, age-related decline in NAD(P)H concentration and redox ratio. With complex I of the electron transport chain inhibited by rotenone, treatment with glutamate or ionomycin only resulted in the increase in NAD(P)H fluorescence. High-performance liquid chromatography analysis of adenine nucleotides in brain extracts showed 50% less nicotinamide adenine dinucleotide (NADH) and almost twice as much oxidized nicotinamide adenine dinucleotide, demonstrating a more oxidized ratio in old than middle-aged brain. Resting glutathione content also declined with age and further decreased with glutamate treatment without accompanying changes in adenosine triphosphate levels. We conclude that age does not affect production of NADH by dehydrogenases but that old-aged neurons consume more NADH and glutathione, leading to a catastrophic decline in redox ratio.

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.

Pattern separation and completion of distinct axonal inputs transmitted via micro-tunnels between co-cultured hippocampal dentate, CA3, CA1 and entorhinal cortex networks.

OBJECTIVE: Functions ascribed to the hippocampal sub-regions for encoding episodic memories include the separation of activity patterns propagated from the entorhinal cortex (EC) into the dentate gyrus (DG) and pattern completion in CA3 region. Since a direct assessment of these functions is lacking at the level of specific axonal inputs, our goal is to directly measure the separation and completion of distinct axonal inputs in engineered pairs of hippocampal sub-regional circuits. APPROACH: We co-cultured EC-DG, DG-CA3, CA3-CA1 or CA1-EC neurons in a two-chamber PDMS device over a micro-electrode array (MEA60), inter-connected via distinct axons that grow through the micro-tunnels between the compartments. Taking advantage of the axonal accessibility, we quantified pattern separation and completion of the evoked activity transmitted through the tunnels from source into target well. Since pattern separation can be inferred when inputs are more correlated than outputs, we first compared the correlations among axonal inputs with those of target somata outputs. We then compared, in an analog approach, the distributions of correlation distances between rate patterns of the axonal inputs inside the tunnels with those of the somata outputs evoked in the target well. Finally, in a digital approach, we measured the spatial population distances between binary patterns of the same axonal inputs and somata outputs. MAIN RESULTS: We found the strongest separation of the propagated axonal inputs when EC was axonally connected to DG, with a decline in separation to CA3 and to CA1 for both rate and digital approaches. Furthermore, the digital approach showed stronger pattern completion in CA3, then CA1 and EC. SIGNIFICANCE: To the best of our knowledge, these are the first direct measures of pattern separation and completion for axonal transmission to the somata target outputs at the rate and digital population levels in each of four stages of the EC-DG-CA3-CA1 circuit.

Active 3-D microscaffold system with fluid perfusion for culturing in vitro neuronal networks.

This work demonstrated the design, fabrication, packaging, and characterization of an active microscaffold system with fluid perfusion/nutrient delivery functionalities for culturing in vitro neuronal networks from dissociated hippocampal rat pup neurons. The active microscaffold consisted of an 8 x 8 array of hollow, microfabricated, SU-8 towers (1.0 mm or 1.5 mm in height), with integrated, horizontal, SU-8 cross-members that connect adjacent towers, thus forming a 3-D grid that is conducive to branching, growth, and increased network formation of dissociated hippocampal neurons. Each microtower in the microscaffold system contained a hollow channel and multiple fluid ports for media delivery and perfusion of nutrients to the in vitro neuronal network growing within the microscaffold system. Additionally, there were two exposed Au electrodes on the outer wall of each microtower at varying heights (with insulated leads running within the microtower walls), which will later allow for integration of electrical stimulation/recording functionalities into the active microscaffold system. However, characterization of the stimulation/recording electrodes was not included in the scope of this paper. Design, fabrication, fluid packaging, and characterization of the active microscaffold system were performed. Furthermore, use of the active microscaffold system was demonstrated by culturing primary hippocampal embryonic rat pup neurons, and characterizing cell viability within the microscaffold system.

Human primary brain tumor cell growth inhibition in serum-free medium optimized for neuron survival.

Glioblastoma is the most common primary brain tumor in adults from which about 15,000 patients die each year in the United States. Despite aggressive surgery, radiotherapy and chemotherapy, median survival remains only 1 year. Here we evaluate growth of primary human brain tumor cells in a defined nutrient culture medium (Neuregen) that was optimized for neuron regeneration. We hypothesized that Neuregen would inhibit tumor cell growth because of its ability to inhibit gliosis in rat brain. Tumor tissue was collected from 18 patients including 10 males and 8 females (mean age 60+/-12 years) who underwent craniotomy for newly diagnosed, histologically confirmed brain tumors. The tissue was shipped overnight in Hibernate transport medium. Tumor cells were isolated and plated in Neurobasal/serum or Neuregen on culture plastic. After 1 week, growth in Neuregen was significantly less in 9/10 glioblastoma multiforme cases, 5/5 meningioma cases and 3/3 cases of brain metastasis. Analysis of deficient formulations of Neuregen and formulations to which selected components were added back implicate no single active component. However, individual cases were sensitive to corticosterone, selenium, ethanolamine, fatty acids and/or antioxidants. Therefore, a defined culture medium that promotes neuron regeneration inhibits the growth of human primary glioblastoma, meningioma and metastatic tumor cells in culture. The possible in vivo efficacy of Neuregen for treatment of brain tumor resections remains to be determined.

Specific CA3 neurons decode neural information of dentate granule cells evoked by paired-pulse stimulation in co-cultured networks.

CA3 and dentate gyrus (DG) neurons are cultured in two-chamber devices on multi-electrode arrays (MEAs) and connected via micro-tunnels. In order to evoke time-locked activity, paired-pulse stimulation is applied to 22 different sites and repeated 25 times in each well in 5 MEA co-cultures and results compared to CA3-CA3 and DG-DG networks homologous controls. In these hippocampal sub-regions, we focus on the mechanisms underpinning a network's ability to decode the identity of site specific stimulation from analysis of evoked network responses using a support vector machine classifier. Our results indicate that a pool of CA3 neurons is able to reliably decode the identity of DG stimulation site information.

Narrow microtunnel technology for the isolation and precise identification of axonal communication among distinct hippocampal subregion networks.

Communication between different sub regions of the hippocampus is fundamental to learning and memory. However accurate knowledge about information transfer between sub regions from access to the activity in individual axons is lacking. MEMS devices with microtunnels connecting two sub networks have begun to approach this problem but the commonly used 10 µm wide tunnels frequently measure signals from multiple axons. To reduce this complexity, we compared polydimethylsiloxane (PDMS) microtunnel devices each with a separate tunnel width of 2.5, 5 or 10 µm bridging two wells aligned over a multi electrode array (MEA). Primary rat neurons were grown in the chambers with neurons from the dentate gyrus on one side and hippocampal CA3 on the other. After 2-3 weeks of culture, spontaneous activity in the axons inside the tunnels was recorded. We report electrophysiological, exploratory data analysis for feature clustering and visual evidence to support the expectation that 2.5 µm wide tunnels have fewer axons per tunnel and therefore more clearly delineated signals than 10 or 5 µm wide tunnels. Several measures indicated that fewer axons per electrode enabled more accurate detection of spikes. A clustering analysis comparing the variations of spike height and width for different tunnel widths revealed tighter clusters representing unique spikes with less height and width variation when measured in narrow tunnels. Wider tunnels tended toward more diffuse clusters from a continuum of spike heights and widths. Standard deviations for multiple cluster measures, such as Average Dissimilarity, Silhouette Value (S) and Separation Factor (average dissimilarity/S value), support a conclusion that 2.5 µm wide tunnels containing fewer axons enable more precise determination of individual action potential peaks, their propagation direction, timing, and information transfer between sub networks.