bioRxiv Subject Collection: Neuroscience's Journal

A Methodology for Specific Disruption of Microtubules in Dendritic Spines
Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

Activating soluble adenylyl cyclase protects mitochondria, rescues retinal ganglion cells, and ameliorates visual dysfunction caused by oxidative stress
Oxidative stress is a key factor causing mitochondrial dysfunction and retinal ganglion cell (RGC) death in glaucomatous neurodegeneration. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway is involved in mitochondrial protection, promoting RGC survival. Soluble adenylyl cyclase (sAC) is one of the key regulators of the cAMP/PKA signaling pathway. However, the precise molecular mechanisms underlying the sAC-mediated signaling pathway and mitochondrial protection in RGCs that counter oxidative stress are not well characterized. Here, we demonstrate that sAC plays a critical role in protecting RGC mitochondria from oxidative stress. Using mouse models of oxidative stress, we found that activating sAC protected RGCs, blocked AMP-activated protein kinase activation, inhibited glial activation, and improved visual function. Moreover, we found that this is the result of preserving mitochondrial dynamics (fusion and fission), promoting mitochondrial bioenergetics and biogenesis, and preventing metabolic stress and apoptotic cell death in a paraquat oxidative stress model. Notably, sAC activation ameliorated mitochondrial dysfunction in RGCs by enhancing mitochondrial biogenesis, preserving mitochondrial structure, and increasing ATP production in oxidatively stressed RGCs. These findings suggest that activating sAC enhances the mitochondrial structure and function in RGCs to counter oxidative stress, consequently promoting RGC protection. We propose that modulation of the sAC-mediated signaling pathway has therapeutic potential acting on RGC mitochondria for treating glaucoma and other retinal diseases.

Astrocytic thrombospondins 1 and 2 are required for cortical synapse development controlling instrumental performance
During development, controlled synaptogenesis is required to form functioning neural circuits that underlie cognition and behavior. Astrocytes, a major glial-cell type in the central nervous system (CNS), promote synapse formation by secreting synaptogenic proteins. Thrombospondins 1 and 2 (TSP1/2), which act through their neuronal receptor 2{delta}-1, are required for proper intracortical excitatory synaptogenesis. In the adult brain, the loss of 2{delta}-1 impairs training-induced excitatory synaptogenesis in the anterior cingulate cortex (ACC), and this impairment leads to increased effort-exertion during high-effort tasks. Here, we tested whether TSP1 and TSP2 are required for controlling effort during operant conditioning by using a lever press for food reward training in mice. Surprisingly, we found that constitutive loss of TSP1/2 significantly reduced lever pressing performance when the effort required for a food reward was increased, a phenotype opposite of 2{delta}-1 loss. Loss of TSP1/2 reduced excitatory synapse number significantly in adult brains. However, in the ACC of TSP1/2 knockout mice, there was still training-induced excitatory synaptogenesis, likely through the upregulation of TSP4, a TSP isoform that is also synaptogenic. Unexpectedly, we also found a significant increase in inhibitory synapse number and function in the ACC of TSP1/2 knockout mice, which was eliminated after training. Finally, we found that astrocyte-specific ablation of TSP1/2 in developing but not adult astrocytes is sufficient to reduce performance during high-effort tasks. Taken together, our study highlights the importance of developmental astrocyte-derived synaptogenic cues TSP1 and 2 in establishing excitatory and inhibitory circuits that control effort during operant conditioning in adults.

Photothrombosis induced cortical stroke produces electrographic epileptic biomarkers in mice
Objective: Interictal epileptiform spikes, high-frequency ripple oscillations, and their co-occurrence (spike ripples) in human scalp or intracranial voltage recordings are well-established epileptic biomarkers. While clinically significant, the neural mechanisms generating these electrographic biomarkers remain unclear. To reduce this knowledge gap, we introduce a novel photothrombotic stroke model in mice that reproduces focal interictal electrographic biomarkers observed in human epilepsy. Methods: We induced a stroke in the motor cortex of C57BL/6 mice unilaterally (N=7) using a photothrombotic procedure previously established in rats. We then implanted intracranial electrodes (2 ipsilateral and 2 contralateral) and obtained intermittent local field potential (LFP) recordings over several weeks in awake, behaving mice. We evaluated the LFP for focal slowing and epileptic biomarkers - spikes, ripples, and spike ripples - using both automated and semi-automated procedures. Results: Delta power (1-4 Hz) was higher in the stroke hemisphere than the non-stroke hemisphere in all mice (p<0.001). Automated detection procedures indicated that compared to the non-stroke hemisphere, the stroke hemisphere had an increased spike ripple (p=0.006) and spike rates (p=0.039), but no change in ripple rate (p=0.98). Expert validation confirmed the observation of elevated spike ripple rates (p=0.008) and a trend of elevated spike rate (p=0.055) in the stroke hemisphere. Interestingly, the validated ripple rate in the stroke hemisphere was higher than the non-stroke hemisphere (p=0.031), highlighting the difficulty of automatically detecting ripples. Finally, using optimal performance thresholds, automatically detected spike ripples classified the stroke hemisphere with the best accuracy (sensitivity 0.94, specificity 0.94). Significance: Cortical photothrombosis-induced stroke in commonly used C57BL/6 mice produces electrographic biomarkers as observed in human epilepsy. This model represents a new translational cortical epilepsy model with a defined irritative zone, which can be broadly applied in transgenic mice for cell type specific analysis of the cellular and circuit mechanisms of pathologic interictal activity.

Repeated Exposure Decreases Aesthetic Chills Likelihood but Increases Intensity
Aesthetic chills are a peak emotional response to affectively charged stimuli such as music, films, or speech. This study investigates the impact of repeated exposure on the frequency and intensity of aesthetic chills. Through a longitudinal approach, we quantified changes in chill likelihood, intensity, and pleasure across multiple exposures, focusing on audio stimuli. Participants (n = 58) were randomly exposed to 6 chill-evoking stimuli pre-validated on the population of interest, in a counterbalanced order. Our findings revealed a significant decrease in the likelihood of experiencing chills with repeated exposure, suggesting habituation to chills itself or potential fatigue in response to aesthetic stimuli. The study also identified distinct demographic and psychophysiological response patterns across different participant groups, indicating variability in chill responses. These results provide insights into the dynamic nature of aesthetic experiences and their underlying neural mechanisms, with implications for understanding emotional and reward processing in psychophysiology.

Dorsal CA1 silencing degrades tactile short-term memory
Previous studies in rodents showed that the hippocampus is involved in spatial short-term memory (STM), but hippocampal necessity for maintaining short-term sensory memories is unknown. Here, we develop tactile discrimination and STM tasks for freely-moving mice. Subjects learn to discriminate between textures after four shaping sessions and a single post-shaping session, and learn the STM task within a dozen sessions. Transient closed-loop silencing of dorsal hippocampal region CA1 during memory maintenance degrades task performance, compared to interleaved control blocks. Thus, uninterrupted hippocampal activity is required for acting upon tactile information maintained in STM. The findings suggest that the role of the hippocampus extends beyond spatial navigation, encoding memories, and long-term consolidation of experiences.

Real-time control of a hearing instrument with EEG-based attention decoding
Restoring normal speech perception in everyday noisy acoustic environments remains an outstanding challenge for hearing aids. Speech separation technology is improving rapidly but hearing instrument technology cannot fully exploit this advance without knowing which sound sources the user wants to hear. Even with high-quality source separation, the hearing aid must know which speech streams to enhance and which to suppress. Advances in EEG-based decoding of auditory attention raise the potential of a neuro-steered hearing instrument that selectively enhances the sound sources that a hearing-impaired listener is focusing their attention on. Here, we present a real-time brain-computer interface (BCI) system implementing this concept. Our system combines a stimulus-response model based on canonical correlation analysis (CCA) for real-time EEG attention decoding with a multi-microphone hardware platform enabling low-latency real-time speech separation through spatial beamforming. In this paper, we provide an overview of the system and its various components and discuss prospects and limitations of the technology. We illustrate its application with case studies of listeners steering acoustic feedback of competing speech streams via real-time attention decoding. A software implementation code of the system is publicly available for further research and explorations.

The Molecular Determinants of a Universal Prion Acceptor
In prion diseases, the species barrier limits the transmission of prions from one species to another. However, cross-species prion transmission is remarkably efficient in bank voles, and this phenomenon can be recapitulated in mice by expression of the bank vole prion protein (BVPrP). The molecular determinants of BVPrP's ability to function as a universal or near-universal acceptor for prions remain incompletely defined. Building on our finding that cultured cells expressing BVPrP can replicate both mouse and hamster prion strains, we conducted a systematic analysis to identify key residues in BVPrP that permit cross-species prion replication. Consistent with previous findings, we demonstrate that residues N155 and N170 of BVPrP, which are absent in mouse PrP but present in hamster PrP, are critical for cross-species prion replication. Additionally, BVPrP residues V112, I139, and M205, which are absent in hamster PrP but present in mouse PrP, are also required to enable replication of both mouse and hamster prions. Unexpectedly, we found that residues E227 and S230 near the C-terminus of BVPrP severely restrict the accumulation of prions following cross-species prion challenge, suggesting that they may have evolved to counteract the inherent propensity of BVPrP to misfold. PrP variants with an enhanced ability to replicate both mouse and hamster prions displayed accelerated spontaneous aggregation kinetics in vitro. These findings suggest that BVPrP's unusual properties are governed by a key set of amino acids and that the enhanced misfolding propensity of BVPrP may enable cross-species prion replication.

Localized estimation of event-related neural source activity from simultaneous MEG-EEG with a recurrent neural network
Estimating intracranial current sources underlying the electromagnetic signals observed from extracranial sensors is a perennial challenge in non-invasive neuroimaging. Established solutions to this inverse problem treat time samples independently without considering the temporal dynamics of event-related brain processes. This paper describes current source estimation from simultaneously recorded magneto- and electro-encephalography (MEEG) using a recurrent neural network (RNN) that learns sequential relationships from neural data. The RNN was trained in two phases: (1) pre-training and (2) transfer learning with L1 regularization applied to the source estimation layer. Performance of using scaled labels derived from MEEG, magnetoencephalography (MEG), or electroencephalography (EEG) were compared, as were results from volumetric source space with free dipole orientation and surface source space with fixed dipole orientation. Exact low-resolution electromagnetic tomography (eLORETA) and mixed-norm L1/L2 (MxNE) source estimation methods were also applied to these data for comparison with the RNN method. The RNN approach outperformed other methods in terms of output signal-to-noise ratio, correlation and mean-squared error metrics evaluated against ground-truth event-related field (ERF) and event-related potential (ERP) waveforms. Using MEEG labels with fixed-orientation surface sources produced the most consistent estimates. To estimate sources of ERF and ERP waveforms, the RNN generates temporal dynamics within its internal computational units, driven by sequential structure in neural data used as training labels. It thus provides a data-driven model of computational transformations from psychophysiological events into corresponding event-related neural signals, which is unique among MEG and EEG source reconstruction solutions.

Generative Adversarial Implicit Successor Representation
We propose a novel method to implicitly encode Successor Representations (SRs) using a Generative Adversarial Network (GAN). SRs are a method to encode states of the environment in terms of their predictive relationships with other states, which can be used to predict long-term future rewards. In standard explicit methods, the value of SR is found from an explicit map between future states after an action or to find an approximate function. Instead, our method encodes SR implicitly using a GAN. The distribution of samples generated by the GAN system approximates the successor representation. We also suggest an action decision procedure for the implicit encoding of SR. The system makes the decision using an analysis-by-synthesis procedure that it attempts to synthesize a sample that can explain the action decision constraints of the current and target states . Our system is different from the classical SR in several points. It can sample actual samples reflecting SR distribution, which is not easy for explicit models. It can also get around the issue of explicitly representing probabilities or successor representation values and doing math over them. We tested our system in a toy environment, where the agent could learn the implicit successor representation successfully and use it for action decisions.

The inflammatory micro-environment induced by targeted CNS radiotherapy is underpinned by disruption of DNA methylation
Although targeted radiotherapy (RT) is integral to the increasing survival of cancer patients, it has significant side-effects, the cellular and molecular mechanisms of which are not fully understood. During RT epigenetic changes occur in neoplastic tissue, but few studies have assessed these in non-neoplastic tissue and results are highly variable. Using bulk DNA methylation and RNA sequencing as well as spatial transcriptomics (ST) in a unique cohort of patient tissue samples, we show distinct differences in DNA methylation patterns in irradiated brain tissue, whilst ST characterisation identifies specific micro-environmental niches present after irradiation and highlights neuropeptides that could be propagating neuroinflammation. We also show that in a cerebral organoid (CO) model of early changes in neurons after irradiation there are similar DNA methylation alterations and disruption of the DNA methylation machinery, suggesting that early but persistent epigenetic dysregulation plays a role in neurotoxicity. We provide a link between radiotherapy induced neuroinflammation and disruption of DNA methylation for the first time and suggest possible driving mechanisms for this chronic neuroinflammation.

Human brain aging heterogeneity observed from multi-region omics data reveals a subtype closely related to Alzheimer's disease
INTRODUCTION: The interconnection between brain aging and Alzheimer's disease (AD) remain to be elucidated. METHODS: We investigated multi-omics (transcriptomics and proteomics) data from multiple brain regions (i.e., the hippocampus (HIPP), prefrontal cortex (PFC), and cerebellum (CRBL)) in cognitively normal individuals. RESULTS: We found that brain samples could be divided into ADL (AD-like) and NL (normal) subtypes which were correlated across brain regions. The differentially expressed genes in the ADL samples highly overlapped with AD gene signatures and the changes were consistent across brain regions (PFC and HIPP) in the multi-omics data. Intriguingly, the ADL subtype in PFC showed more differentially expressed genes than other brain regions, which could be explained by the baseline gene expression differences in the PFC NL samples. DISCUSSION: We conclude that brain aging heterogeneity widely exists, and our findings corroborate with the hypothesis that AD-related changes occur decades before the clinical manifestation of cognitive impairment in a sub-population.

Electrocorticographic and Astrocytic Signatures of Stearoyl-CoA Desaturase Inhibition in the Triple Transgenic Mouse Model of Alzheimer's Disease
The symptomatology of Alzheimer's disease (AD) includes cognitive deficits and sleep disturbances. Recent findings suggest the involvement of dysfunctions in lipid metabolism, such as oleic acid build-up, in the brain of AD patients and animal models. In addition, the inhibition of stearoyl-CoA desaturase (SCD), a lipid-converting enzyme, was shown to restore memory in triple transgenic (3xTg)-AD mice. In the brain, astrocytes regulate the synthesis of specific lipids. Alterations in astrocytes and their function were reported in AD patients and animal models, and astrocytes have been implicated in the regulation of sleep. However, the relationship between sleep disturbances, astrocytes and lipid metabolism remains to be explored in AD. This project thus aimed at assessing whether the inhibition of SCD restores sleep in 3xTg-AD mice, and whether this associated with modifications in astrocytic function. Wild-type (WT) and 3xTg-AD female mice (4-months old) received intracerebroventricular infusion of a SCD inhibitor (SCDi) or vehicle for 28 days, and a 24-hour electrocorticographic (ECoG) recording was conducted post-treatment. Post-mortem brain slices were stained for the astrocytic markers glial fibrillary acidic protein (GFAP) and 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) to perform cell counting and/or morphological evaluation in the hippocampus, lateral hypothalamus and thalamus. The results indicate that the reduced time spent awake and increased time spent in slow wave sleep (SWS) in 3xTg-AD mice was not restored by the SCDi treatment. Similar observations were made concerning the increased number of wake and SWS bouts in 3xTg-AD mice. Rhythmic and scale-free ECoG activity were markedly altered in 3xTg-AD mice for all wake/sleep states, and SCDi significantly altered these phenotypes in a different manner in mutant mice in comparison to WT mice. GFAP- and ALDH1L1-positive cell densities were elevated in the hippocampus and lateral hypothalamus/thalamus of 3x-Tg-AD mice, respectively, and SCDi rescued the increase in the CA1 region in particular. Overall, these findings suggest that the multiple wake/sleep alterations in 3xTg-AD mice are not substantially restored by targeting lipid metabolism using SCD inhibition, at least for the targeted age window, but that this treatment can revert hippocampal changes in astrocytes. This work will benefit the understanding of the pathophysiology related to AD and associated sleep disturbances.

Temporal dynamics of human color processing measured using a continuous tracking task
We characterized the temporal dynamics of color processing using a continuous tracking paradigm by estimating temporal impulse response functions associated with tracking chromatic Gabor patches. We measured how the lag of these functions changes as a function of chromatic direction and contrast for stimuli in the LS cone contrast plane. In the same set of subjects, we also measured detection thresholds for stimuli with matched spatial, temporal, and chromatic properties. We created a model of tracking and detection performance to test if a common representation of chromatic contrast accounts for both measures. The model summarizes the effect of chromatic contrast over different chromatic directions through elliptical isoresponse contours, the shapes of which are contrast independent. The fitted elliptical isoresponse contours have essentially the same orientation in the detection and tracking tasks. For the tracking task, however, there is a striking reduction in sensitivity to signals originating in the S cones. The results are consistent with common chromatic mechanisms mediating performance on the two tasks, but with task-dependent relative weighting of signals from L and S cones.

The spiking output of the mouse olfactory bulb encodes large-scale temporal features of natural odor environments
Spatiotemporal dynamics of natural odor environment have informative features for animals navigating to an odor source. Population activity in the olfactory bulb (OB) has been shown to follow plume dynamics to a moderate degree (Lewis et al., 2021), but it is unknown whether the ability to follow plume dynamics is driven by individual cells or whether it emerges at the population level. Previous research has explored the responses of individual OB cells to isolated features of plumes, but it is difficult to adequately sample these features as it is still undetermined which features navigating mice employ during olfactory guided search. Here we released odor from an upwind odor source and simultaneously recorded both odor concentration dynamics and cellular response dynamics in awake, head-fixed mice. We found that longer timescale features of odor concentration dynamics were encoded at both the cellular and population level. At the cellular level, plume onset was encoded across all trials and plume offset was encoded for high concentration odors, but not low concentration odors. Although cellular level tracking of plume dynamics was observed to be weak, we found that at the population level, OB activity distinguished whiffs and blanks (accurately detected odor presence versus absence) throughout the duration of a plume. Even ~20 OB cells were enough to accurately encode these features. Our findings indicate that the full range of odor concentration dynamics and high frequency fluctuations are not encoded by OB spiking activity. Instead, relatively lower-frequency dynamics of plumes, such as plume onset, plume offset, whiffs, and blanks, are represented in the OB.

Calcium-dependent regulation of neuronal excitability is rescued in Fragile X Syndrome by a tat-conjugated N-terminal fragment of FMRP
Fragile X Syndrome arises from the loss of Fragile X Messenger Ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber LTP by adjusting Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knockout (KO) tsA-201 cells and cerebellar sections from Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex, and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signalling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signalling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of Fragile X Syndrome.

A comprehensive investigation of intracortical and corticothalamic models of alpha rhythms
Alpha rhythms are a robust phenomenon prominently observed in posterior resting state electroencephalogram (EEG) that has been shown to play a key role in a number of cognitive processes. However, the underlying mechanisms behind their generation is poorly understood. Here, we showcase the most concrete, mathematically-expressed theoretical foundations for understanding the neural mechanisms underlying the alpha rhythmogenesis. The neural population models of interest are Jansen-Rit (JR), Moran-David-Friston (MDF), Robinson-Rennie-Wright (RRW), and Liley-Wright (LW). Common elements between all models are identified, such as the description of each neural population in the form of a second-order differential equation with a potential-to-rate operator represented as a sigmoid and a rate-to-potential operator usually expressed as an impulse response. Even though these models have major differences, they can be meaningfully compared by associating parameters of analogous biological significance, which we summarize with a unified parameter table. With these correspondences, rate constants and connectivity parameter space is explored to identify common patterns between similar behaviors, such as the role of excitatory-inhibitory interactions in the generation of oscillations. Through stability analysis, two different alpha generation mechanisms were identified: one noise-driven and one self-sustaining oscillation in the form of a limit cycle emerging due to a Andronov-Hopf bifurcation. This work contributes to improving our mechanistic and theoretical understanding on candidate theories of alpha rhythmogenesis.

Artificial neural network for brain-machine interface consistently produces more naturalistic finger movements than linear methods
Brain-machine interfaces (BMI) aim to restore function to persons living with spinal cord injuries by "decoding" neural signals into behavior. Recently, nonlinear BMI decoders have outperformed previous state-of-the-art linear decoders, but few studies have investigated what specific improvements these nonlinear approaches provide. In this study, we compare how temporally convolved feedforward neural networks (tcFNNs) and linear approaches predict individuated finger movements in open and closed-loop settings. We show that nonlinear decoders generate more naturalistic movements, producing distributions of velocities 85.3% closer to true hand control than linear decoders. Addressing concerns that neural networks may come to inconsistent solutions, we find that regularization techniques improve the consistency of tcFNN convergence by 194.6%, along with improving average performance, and training speed. Finally, we show that tcFNN can leverage training data from multiple task variations to improve generalization. The results of this study show that nonlinear methods produce more naturalistic movements and show potential for generalizing over less constrained tasks.