Gregory J. Brewer

Objective. Local field potentials (LFPs) measured in the extracellular matrix of the brain are postulated to arise from the integration of synaptic ionic currents and spread by volume conduction. However, there is a lack of consensus on whether these spatiotemporal voltage gradients are just an epiphenomenon of spiking or if the LFPs play a functional role in information processing. Approach. To examine a potential functional role of LFPs in information processing, we developed a microfluidic device that allows neurons from the hippocampal formation to self-wire through microfluidic channels, effectively isolating the activity of single axons between subregions of the network. Results. We recorded spontaneous theta-band activity (4–10 Hz) in these axons whose power spectra were independent of simultaneous spiking activity. The highest theta amplitudes above noise were seen intermittently in a sparse set of axons from the CA3 into the CA1. Source neurons for the axonal theta were identified through cross correlation. Functionally, sparse axonal theta phase and amplitude correlated with target subregional spiking and more strongly with burst length. Significance. These results suggest that theta voltage oscillations in axons may contribute to activation of slow voltage-gated calcium channels to drive stronger synaptic release of transmitter to coordinate hippocampal activity between subregions. We propose that theta oscillations are controlled by specific ion channels distinct from those that generate spikes, a multiplex coding mechanism for inter-regional communication with implications for routing, executive control, electroencephalogram, disease states, and artificial neural networks.

Local field potentials (LFPs) measured in the extracellular matrix of the brain are postulated to arise from the integration of synaptic ionic currents and spread by volume conduction. However, there is a lack of consensus on whether these spatiotemporal voltage gradients are just an epiphenomenon of spiking or if the LFPs play a functional role in information processing. To examine a potential functional role of LFPs in information processing, we developed a microfluidic device that allows neurons from the hippocampal formation to self-wire through microfluidic channels, effectively isolating the activity of single axons between subregions of the network. We recorded spontaneous theta-band activity (4-10 Hz) in these axons whose power spectra were independent of simultaneous spiking activity. A sparse set of axons from the CA3 into the CA1 had the highest theta amplitudes. Source neurons for the axonal theta were identified through cross correlation. Functionally, sparse axonal theta phase and amplitude correlated with target subregional spiking and more strongly with burst length. These results suggest that theta voltage oscillations in axons may contribute to activation of slow voltage-gated calcium channels to drive stronger synaptic release of transmitter to coordinate hippocampal activity between subregions. We propose that theta oscillations are controlled by specific ion channels distinct from those that generate spikes, a multiplex coding mechanism for inter-regional communication with implications for routing, executive control, disease states and artificial neural networks.

Age-related declines in neuronal bioenergetic levels may limit vesicular trafficking and autophagic clearance of damaged organelles and proteins. Age-related ATP depletion would impact cognition dependent on ionic homeostasis, but limits on proteostasis powered by GTP are less clear. We used neurons isolated from aged 3xTg-AD Alzheimer’s model mice and a novel genetically encoded fluorescent GTP sensor (GEVAL) to evaluate live GTP levels in situ. We report an age-dependent reduction in ratiometric measurements of free/bound GTP levels in living hippocampal neurons. Free GTP colocalized in the mitochondria decreased with age accompanied by the accumulation of free GTP-labeled vesicular structures. The energy dependence of autophagy was demonstrated by depletion of GTP with rapamycin stimulation, while bafilomycin inhibition of autophagy raised GTP levels. Twenty-four-hour supplementation of aged neurons with the NAD precursor nicotinamide and the Nrf2 redox modulator EGCG restored GTP levels to youthful levels and mobilized endocytosis and lysosomal consumption for autophagy via the respective GTPases Rab7 and Arl8b. This vesicular mobilization promoted the clearance of intraneuronal Aβ aggregates, improved viability, and lowered protein oxidative nitration in AD model neurons. Our results reveal age- and AD-related neuronal GTP energy deficits that impair autophagy and endocytosis. GTP deficits were remediated by an external NAD precursor together with a Nrf2 redox modulator which suggests a translational path. Graphical Abstract

Each sub-region of the hippocampus plays a critical computational role in the formation of episodic learning and memory, but studies have yet to show and interpret the individual spiking dynamics of each region and how that information is passed between each subregion. This is in part due to the difficulty in accessing individual communicating axons. Here, we created a novel microfluidic device that facilitates network growth of four separated hippocampal subregions over a micro-electrode array. This device enabled monitoring single axons over two electrodes so direction of spike propagation in interregional communication could be ascertained. In this in vitro hippocampal study, we compared spiking dynamics across two novel four-compartment device architectures: one with four sets of axon tunnels between subregions that excluded the perforant pathway from EC-CA3, and one with five sets of axon tunnels that included the EC-CA3 connection. We found 30–90% faster feed-forward firing rates (shorter interspike intervals) in axons in the five-tunnel model with 35–75% slower bursting dynamics (longer interburst intervals) compared to the four-tunnel model. The CA3-CA1 and CA1-EC axons had more spikes in bursts in the five-tunnel architecture than the four-tunnel counterpart suggesting more structured information transfer. Feedback firing rates were similar between configurations. The faster feed-forward inter-regional spiking in the more natural five-tunnel than the four-tunnel configuration suggests tighter control of spiking and possibly more precise communication between subregions.

Lipid homeostasis plays a crucial role in neuronal function, yet its dynamics during aging and neurodegenerative diseases remain poorly understood. Our study unveils critical age-related changes in lipid polarity and lipid droplet characteristics in hippocampal neurons from non-transgenic (NTg) mice and from an Alzheimer's disease-model (3xTg-AD). Using advanced spectral imaging and phasor analysis techniques, we tracked lipid polarity with Nile Red in vitro across various cellular compartments and quantified lipid droplet features. We discovered that NTg neurons exhibit a progressive increase in global lipid polarity from young to middle age, followed by a slight decrease in old age. This pattern suggests that neurons actively regulate their lipid composition throughout the lifespan, potentially in response to changing cellular needs. In contrast, AD-like (or 3xTg-AD) neurons fail to show this age-related increase in lipid polarity, instead displaying a consistent reduction in lipid polarity across all ages. Lipid droplet analysis revealed a transient accumulation of larger droplets in middle-aged NTg neurons, while AD-transgenic neurons showed early and persistent increases in lipid droplet size and number. Principal component analysis uncovered coordinated changes in lipid polarity and droplet characteristics, highlighting distinct patterns of lipid partitioning in NTg and AD-affected neurons. These findings suggest that AD-associated genetic modifications disrupt normal age-related adaptations in lipid metabolism and organization. Our results provide new insights into the complex interplay between lipid homeostasis, aging, and AD-genotypic stress. Understanding these dynamics may open new avenues for developing therapeutic strategies to maintain neuronal health and potentially slow AD progression.

Age-related declines in neuronal bioenergetic levels may limit vesicular trafficking and autophagic clearance of damaged organelles and proteins. Age-related ATP depletion would impact cognition dependent on ionic homeostasis, but limits on proteostasis powered by GTP are less clear. We used neurons isolated from aged 3xTg-AD Alzheimer’s model mice and a novel genetically encoded fluorescent GTP sensor (GEVAL) to evaluate live GTP levels in situ. We report an age-dependent reduction in ratiometric measurements of free/bound GTP levels in living hippocampal neurons. Free-GTP co-localized in the mitochondria decreased with age accompanied by the accumulation of free-GTP labeled vesicular structures. The energy dependence of autophagy was demonstrated by depletion of GTP with rapamycin stimulation, while bafilomycin inhibition of autophagy raised GTP levels. 24 hr. supplementation of aged neurons with the NAD precursor nicotinamide and the Nrf2 redox modulator EGCG restored GTP levels to youthful levels and mobilized endocytosis and lysosomal consumption for autophagy via the respective GTPases Rab7 and Arl8b. This vesicular mobilization promoted the clearance of intraneuronal Aβ aggregates and lowered protein oxidative nitration in AD model neurons. Our results reveal age– and AD-related neuronal GTP energy deficits that impair autophagy and endocytosis. GTP deficits were remediated by an external NAD precursor together with a Nrf2 redox modulator which suggests a translational path.

Background: Nicotinamide, a form of B3 vitamin, is an NAD⁺ precursor that reduces pTau₂₃₁ levels via histone deacetylase inhibition in murine models of Alzheimer’s disease (AD). A recent phase 2a randomized placebo-controlled trial tested high-dose oral nicotinamide for the treatment of early AD. While nicotinamide demonstrated good safety and tolerability, it did not significantly lower CSF pTau₂₃₁, the primary biomarker endpoint of the study. Characterization of nicotinamide’s pharmacokinetics and metabolites in the blood and CSF is needed. Methods: In these post hoc, blinded analyses of plasma and CSF samples from the completed two-site placebo controlled randomized trial testing of 1500 mg PO BID oral nicotinamide, we used mass spectroscopy to measure nicotinamide and its inactive metabolite 1-methyl-nicotinamide in plasma at baseline, 6, and 12 months and in CSF at baseline and 12 months from 23 participants on drug and 24 on placebo. Results: Pharmacokinetic analysis found mean 12 month plasma nicotinamide increased > 130-fold to 52 μM while mean methyl-nicotinamide increased > 600-fold to 91 μM in individuals receiving nicotinamide compared to those receiving placebo, whose levels were unchanged from baseline. However, CSF nicotinamide was only measurable in 6 of the 19 available participants (32%) (mean increase of at least 147-fold to 18 μM). These CSF nicotinamide concentrations were 66% of their plasma levels, indicating good CNS bioavailability in only some participants. In contrast to CSF nicotinamide, more treated participants had higher CSF methyl-nicotinamide (n = 9, 43 μM), suggesting high-dosage nicotinamide was sufficient to pass the blood–brain barrier, but 13 of 19 were metabolically inactivated. Treatment favorably decreased mean pTau₂₃₁ levels by 34% in those six participants with elevated CSF levels of nicotinamide, compared to 3% elevation in participants who did not have elevated CSF nicotinamide, and a 3% decrease for placebo. No such relationships were observed for total tau, pTau₁₈₁, or amyloid beta biomarkers. Conclusions: Our findings suggest that oral administration markedly increased mean plasma nicotinamide levels, however CSF levels were below quantitation in a majority of participants and there was extensive metabolic inactivation to methyl-nicotinamide. Both the bioavailability and rapid metabolic methylation need to be addressed if nicotinamide is further developed as a potential intervention for AD. Trial registration: NCT03061474, last updated 2023–10-17. https://clinicaltrials.gov/study/NCT03061474.

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.

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.