Communication between different sub regions of the hippocampus is fundamental to learning and memory. However accurate knowledge about information transfer between sub regions in individual axons is lacking. MEMS devices with microtunnels connecting two sub networks have begun to approach this problem but common 10 µm wide tunnels frequently produce ambiguous interfering spikes 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). After 2-3 weeks of culturing primary rat neurons, with dentate gyrus on one side and hippocampal CA3 on the other of the chamber, spontaneous activity in the axons inside the tunnels was recorded. We report electrophysiological, exploratory data analysis for feature clustering and visual evidence to support our expectation that compared to 10 µm wide tunnels, 2.5 µm wide tunnels have fewer axons per tunnel. Clustering measures comparing the variations of spike height and width for different tunnel widths revealed tighter clusters of spikes with less height and width variation for 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 Derivative (S) and Separation Factor, support a conclusion that 2.5 μm wide tunnels with fewer axons enable more precise determinations of individual action potential peaks, their propagation direction and hence timing of information transfer between sub networks.

Aging increases the risk of neurodegenerative diseases including Alzheimer’s disease (AD) and Parkinson’s Disease. AD is a progressive neurodegeneration with loss of cognitive memory, leading to dementia. The goal of this work is to systematically trace an upstream metabolic or energetic shift in aging and AD to identify targets to delay or even reverse the course of aging and pathologies in AD. Based on the epigenetic oxidative redox shift (EORS) theory of aging, we developed the hypothesis that age- and AD-related oxidative shifts deplete NADH levels in hippocampal neurons, which further trigger the downstream energetic and metabolic shifts sensed at NAD+/NADH redox sensitive sites. Regulated by redox-sensitive transcription factors, metabolic shifts are enforced epigenetically, which initiate a vicious cycle and exacerbate the course of aging and AD.

This work investigates upstream oxidative shifts in NADH redox states and verifies causative links to systematic downstream metabolic and energetic networks. As nicotinamide adenine dinucleotide (NAD and NADH) plays critical roles in metabolic oxidation-reduction reactions in energetic pathways as well as redox detoxification systems, we further examined if these age-related oxidized free NADH redox states and depletion of NADH are reversible and restored with external redox modification in primary cultures by manipulating different redox potentials of Cysteine/Cystine (Cys/CySS). NADH measurements were performed on live neurons of primary hippocampal cultures of non-transgenic (NTg) and triple-transgenic AD mouse model (3xTg-AD) across the age-span, by using the fluorescence lifetime imaging microscopy (FLIM) technique. This FLIM technique is a non-invasive approach of the intrinsic fluorescence in NADH. After transformation of pixels of FLIM images by Fast Fourier Transform (FFT), we were able to distinguish different NADH lifetimes, analyze and quantify free and enzyme-bound NADH in subcellular compartments in the phasor plot. We found a strong age effect that diminished the free NADH levels in mitochondria, cytoplasm and nuclei with further depletion with AD genetic load. With an imposed oxidized Cys/CySS state of 0 mV in neuron culture, we found a lower capacity for maintaining free NADH in old age versus young age neurons of both NTg and 3xTg-AD mouse neurons. Remarkably, under an external imposed Cys/CySS reductive state, the mitochondrial free NADH levels were rejuvenated to the levels of young age neurons in both NTg and 3xTg-AD mouse neurons. Our findings suggest a potential reductive treatment to reverse the loss of free NADH in old and AD neurons.

To test our hypothesis of an age-related metabolic shift triggered by oxidation in NADH redox states, we applied untargeted Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to globally detect and measure the metabolite levels from NTg and 3xTg-AD mouse hippocampal brains across the age-span. We observed strong age-related global elevations up to 2-3-fold in energy metabolism including glycolytic ATP-produced steps, fatty acid b-oxidation and branched chain amino acid metabolism. The direction of metabolic changes with age pivoted at NAD+/NADH redox sensitive dehydrogenases. We found for the first-time age-related decreases in the upstream metabolites of dehydrogenases and increases in the downstream metabolites of glycolysis and the TCA cycle. Our results support the hypothesis and provide a potential reductive treatment to possibly counter AD and extend healthy lifespan, by reversal of low free NADH levels in old and Alzheimer’s neurons via a series of NADH-sensor mechanisms.

The hippocampus has long been known to play a central role in various behavioral and cognitive functions. Despite extensive behavioral, anatomical studies postulating roles of the hippocampus and its sub-regions, the underlying dynamics of the neural circuits responsible for region-specific roles in the hippocampus remains poorly understood. In addition, the communication between the hippocampal sub-regions also plays a major role in learning and memory but has not been explored as much due to the lack of access to the individual axons. This paper seeks to understand the role of slow waves (4-100 Hz) known as Local Field Potentials (LFP) in the hippocampal sub-regions, their dynamics and role in information transmission to bind cell assemblies through synchrony by timed-bursts and individual spikes, by space (EC, DG, CA3, CA1) in the hippocampal sub-regions and by frequency (4-11 Hz theta and 30-100 Hz gamma bands). We used a four-chambered polydimethysiloxane (PDMS) micro-tunnel device over a multi-electrode array (MEA) with the engineered living reconstruction of the hippocampal sub-regions. The 5 x 10 μm micro-tunnels between each chamber allow axonal growth and inhibit the migration of 15 μm diameter cell bodies or traversal by dendrites providing insight into the role of axons in inter-regional communication. A significant advantage of our system is the ability to separate activity in the axons in the tunnels from the computations of the neural cells in the wells of the 4-chambered device. Functional connectivity within hippocampal sub-regions and the axonal tunnels helped in ascertaining the role of LFP in information processing and transmission. A computationally simple and linear method, Cross-Correlation of the theta power and the cross correlation of the gamma power was used to estimate synchrony of LFP. Comparison between the theta-theta and gamma-gamma LPF’s from the different sub-regions indicates that the CA3 has the strongest correlation. CA3 sub-region is characterized by recurrent network wiring pattern and such strong correlation can be associated with this kind of network pattern. The spatial distribution of the theta and gamma correlation between electrodes was estimated by computing the correlation as a function of the inter-electrode distance within each sub-region. A common relationship of LFP power with the inverse square of distance during bursting events suggested LFP transmission through synapses of branching dendritic trees in the plane of the network. These first measures of LFP’s in axons suggests axonal transmission of slow waves, independent of spikes and synapses. Understanding these network properties within the hippocampus could provide important information for steps in memory formation, neuromorphic computing and artificial intelligence systems in addition to better detection of hippocampal diseases such as epilepsy and Alzheimer’s disease.

The NEAT trial of high-dose oral nicotinamide efficacy to treat Alzheimer’s Disease ended without reaching the primary endpoint of lowering CSF pTau181. However, knowledge of drug levels in the blood or whether the drug reached the CNS were not included in the primary study. In post hoc, blinded analysis of plasma and CSF samples by mass spectroscopy, we measured blood plasma and CSF levels of nicotinamide and its breakdown product 1-methyl-nicotinamide from 23 patients on drug and 24 patients on placebo for 12 months. We found that mean plasma nicotinamide from drug-treated patient’s increased >130-fold while mean methyl-nicotinamide increased >600-fold over pre-treatment and placebo. Plasma nicotinamide reached the CNS at measurable levels in only 29% of the patients (mean increase of 147-fold), and associated with elevated methyl-nicotinamide in the CSF for 45% of the drug treated patients. Importantly, treatment favorably decreased the concentration of r pTau231 by a mean of 33% in patients with elevated CSF levels of nicotinamide in the CNS compared to 3.7% in those without measurable CSF levels. Our findings suggest nicotinamide may be efficacious if it reaches measurable levels in CSF, but that there are CNS bioavailability problems that include methyl inactivation that need to be addressed in future formulations for treating AD.