bioRxiv Subject Collection: Neuroscience's Journal

Mobile Human Brain Imaging using Functional Ultrasound
Imagine being able to study the human brain in real-world scenarios while the subject displays natural behaviors such as locomotion, social interaction, or spatial navigation. The advent of ultrafast ultrasound imaging brings us closer to this goal with functional Ultrasound imaging (fUSi), a new mobile neuroimaging technique. Here, we present real-time fUSi monitoring of brain activity during walking in a subject with a clinically approved sonolucent skull implant. Our approach utilizes personalized 3D-printed fUSi-helmets for stability, optical tracking for cross-modal validation with fMRI, advanced signal processing to estimate hemodynamic responses and facial tracking of a lick licking paradigm. These combined efforts allowed us to show consistent fUSi signals over 20 months, even during high-motion activities like walking. These results demonstrate the feasibility of fUSi for monitoring brain activity in real-world contexts, marking an important milestone for fUSi-based insights in clinical and neuroscientific research.

Competition between glycine and GABAA receptors for gephyrin controls their equilibrium populations at inhibitory synapses
Glycine and GABA receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission throughout the central nervous system. The receptors co-localise widely at inhibitory synapses in the spinal cord and in the brainstem due to their interaction with an overlapping binding site of the synaptic scaffold protein gephyrin, pointing to a direct competition between the different receptor types. We have put this hypothesis to the test using single molecule approaches to measure receptor-gephyrin interactions in cells and in vitro. We explored the effects of receptor competition at inhibitory synapses in living neurons by measuring the change in the accumulation and effective stabilisation energy of glycine receptors in the presence of interfering GABA receptor complexes through single molecule tracking and diffusion analysis. Secondly, using molecular tweezers, we quantified the thermodynamic properties of receptor-gephyrin binding, demonstrating direct and reversible competition through the addition of interacting peptides in solution. The relatively low affinity of GABA receptor subunits for gephyrin compared to the glycine receptor raises interesting questions about the role of this competition in synaptic plasticity. We hypothesize that GABA and glycine receptor competition constitutes a molecular system designed to reconcile synapse stability and plasticity at mixed inhibitory synapses.

Cannabinoid inhibition of mechanosensitive K+ channels
Cannabidiol (CBD) is a prominent non-psychoactive small molecule produced by cannabis plants used clinically as an antiepileptic. Here, we show CBD and other cannabinoids are potent inhibitors of mechanosensitive two-pore domain K+ (K2P) channels, including TRAAK and TREK-1 that contribute to spike propagation in myelinated axons. Five TRAAK mutations that cause epilepsy or the neurodevelopmental syndrome FHEIG (facial dysmorphism, hypertrichosis, epilepsy, intellectual/developmental delay, and gingival overgrowth) retain sensitivity to cannabinoid inhibition. A cryo-EM structure reveals CBD binds in the intracellular cavity of TREK-1 to sterically block ion conduction. These results show that cannabinoids and endogenous lipids compete for a common binding site to inhibit channel activity, identify mechanosensitive K2Ps as potential physiological targets of CBD, and suggest cannabinoids could counter gain-of-function in TRAAK channelopathies.

Multi-omic integration with human DRG proteomics highlights TNFα signalling as a relevant sexually dimorphic pathway
The peripheral nervous system has been widely implicated in pathological conditions that exhibit distinct clinical presentations in men and women, most notably in chronic pain disorders. Here, we explored this sexual dimorphism at a molecular level. We expanded the available omics landscape in the PNS to include quantitative proteomics of the human dorsal root ganglia (hDRG) and nerve. Using data-independent acquisition mass spectrometry, we uncovered an extensive protein landscape, validated against tissue-specific differences between the nerve and hDRG. Using a combination of multi-omic analyses and in vitro functional support, we then examined sex-differences, highlighting TNF signalling as a relevant sexually dimorphic pathway in males. These results support a functional sexually dimorphism in the periphery, which is of particular importance to sensory- and pain-related clinical translation.

The effect of illumination cues on color constancy in simultaneous identification of illumination and reflectance changes
To provide a stable percept of the surface color of objects, the visual system needs to account for variation in illumination chromaticity. This ability is called color constancy. The details of how the visual system disambiguates effects of illumination and reflectance on the light reaching the eye are still unclear. Here we asked how independent illumination and reflectance judgments are of each other, whether color constancy depends on explicitly identifying the illumination chromaticity, and what kinds of contextual cues support this identification. We studied the simultaneous identification of illumination and reflectance changes with realistically rendered, abstract 3D-scenes. Observers were tasked to identify both of these changes between sequentially presented stimuli. The stimuli included a central object whose reflectance could vary, and a background that only varied due to changes in illumination chromaticity. We manipulated the visual cues available in the background: local contrast and specular highlights. Identification of illumination and reflectance changes was not independent: While reflectance changes were rarely mis-identified as illumination changes, illumination changes clearly biased reflectance judgments. However, correct identification of reflectance changes was also not fully dependent on correctly identifying the illumination change: Only when there was no illumination change in the stimulus did it lead to better color constancy, that is, correctly identifying the reflectance change. Discriminability of illumination changes did not vary based on available visual cues, but discriminability of reflectance changes was improved with local contrast, and to a lesser extent with specular highlights, in the stimulus. We conclude that a failure of color constancy does not depend on a failure to identify illumination changes, but additional visual cues still improve color constancy through better disambiguation of illumination and reflectance changes.

Brain-body circuit by which midbrain dopamine neuronal activity modulates splenic immunity
Midbrain dopamine neurons are integral to central nervous system function, yet their influence on peripheral immunity remains underexplored. This study identifies a neural circuit connecting midbrain dopamine neurons to the spleen through the dorsal vagal complex (DVC) and celiac ganglion. Using DAT:Cre mice and Cre-dependent viral tracers, we demonstrated that midbrain dopamine neurons project to the DVC, where they form synapses and modulate neuronal activity. Electrophysiological recordings revealed that DVC neurons express D1-like and D2-like dopamine receptors, responding to dopamine with altered excitability. Chemogenetic activation of midbrain dopamine neurons induced dopamine release in the DVC and increased cFos expression in both the DVC and celiac ganglion, indicating enhanced neuronal activity along this circuit. Anterograde multi-transsynaptic tracing confirmed connectivity from the midbrain to the spleen via the celiac ganglion. Activation of this circuit resulted in reduced spleen weight as well as a significant decrease in naive CD4+ T-cell populations without affecting total T-cell numbers. These findings unveil a functional midbrain-DVC-celiac ganglion-spleen pathway, through which midbrain dopamine neurons modulate splenic immunity. This novel insight into the neural regulation of the immune system has important implications for diseases involving altered dopamine neurotransmission, highlighting potential targets for immunotherapeutic interventions.

Hormetic curve of dietary mono- and disaccharide content determines weight gain, gut microbiota composition and cognitive ability in mice
Hormesis is defined as dose response phenomenon characterized by low-dose stimulation and high-dose inhibition (Calabrese & Mattson, 2017). To date, low doses of several stressors (intermittent fasting, caloric restriction or selected phytochemicals) have been shown to exert beneficial effects on health (Martin et al., 2006). In the present study, we aimed to determine hormetic factors in a series of diets used in mice. We found that animals fed high-sugar diet (HSD) or high-fat diet (HFD) containing relatively high amounts of mono- and disaccharides become obese compared to animals fed standard diet (STAND) or ketogenic diet (KD) containing low doses of these compounds. Underlying the observed metabolic phenotype may be changes in the composition of the intestinal microbiota, showing u-shaped features in selected species. It is noteworthy that a short-term dietary regimen of several weeks resulted in difficulties in achieving effective scores on a complex cognitive test based on spatial procedural acquisition in the HSD and HFD groups. Our data identify dietary mono- and disaccharide content (commonly known as sugars) as a critical hormetic factor with beneficial/harmful effects at multiple levels of body function.

The impact of sex differences on perceived pain intensity in pain protocol standardization
Background: Sex differences have been widely demonstrated in both acute and chronic pain. Sex differences may have wider impact on research design and analysis than already established. This study addresses an important methodological aspect with regards to how sex differences could influence the design of standardized experimental pain protocols used to characterize an individuals pain response. Methods: This study addresses sex differences of perceived pain at tonic heat pain threshold (HPT). Participants used a computerized visual analogue scale (CoVAS) to continually rated subjective pain intensity during tonic HPT. Metrics (Mean, Standard Deviation, Maximum) were extracted from the CoVAS to characterize perceived pain. Results: Female participants rated pain intensity at HPT significantly lower than male participants across all extracted metrics used to characterize the coVAS rating. The effect of sex on the mean and standard deviation of pain intensity ratings at HPT was medium, while the effect size of sex on the maximum pain intensity rating at HPT was large. Conclusion: The significant sex differences in perceived pain intensity at HPT indicates that methods of standardization to a specific pain intensity merit sex-specific consideration. Additionally, these observed sex differences underscore the necessity for sex specific design across both pre-clinical and clinical studies of pain.

Dysfunctional oscillatory bursting patterns underlie working memory deficits in adolescents with ADHD
Identifying neural markers of clinical symptom fluctuations is prerequisite to developing more precise brain-targeted treatments in psychiatry. We have recently shown that working memory (WM) in healthy adults is dependent on the rise and fall interplay between alpha/beta and gamma bursts within frontoparietal regions, and that deviations in these patterns lead to WM performance errors. However, it is not known whether such bursting deviations underlie clinically relevant WM-related symptoms or clinical status in individuals with WM deficits. In adolescents (n=27) with attention deficit hyperactivity disorder (ADHD), we investigated WM-related dynamics between alpha/beta and gamma bursts in relation to clinical status fluctuations. Participants repeatedly completed a visual Sternberg spatial working memory task during EEG recording as part of their participation in two research studies (n=224 person-sessions). Source localizing EEG data to each structural MRI, the rate and volume of alpha, beta, and gamma bursts were examined within the dorsolateral prefrontal cortex (DLPFC) and posterior parietal cortex (PPC). Alpha/beta and gamma bursts at the DLPFC and PPC displayed complimentary roles in WM processes. Alpha/beta bursting decreased during stimuli encoding and increased during the delay, while gamma bursting was elevated during encoding and decreased during the delay. Deviations in bursting patterns were associated with WM errors and clinical symptoms. We conclude that dysfunctional alpha/beta and gamma burst dynamics within the frontoparietal region underlie both intra-individual WM performance and WM symptom fluctuations in adolescents with ADHD. Such burst dynamics reflect a novel target and biomarker for WM-related treatment development.

Spatial organization of multisensory convergence in mouse isocortex
The diverse functions of different cortical areas are thought to arise from their distinct groups of inputs. However, additional organizing principles may exist in the spatial structure of converging inputs. We investigated spatial convergence patterns of projections from primary sensory areas to other areas throughout the mouse isocortex. We used a large tract tracing dataset to estimate the dimension of the space into which topographical connections from multiple modalities converged within each other cortical area. We call this measure the topography dimension (TD). TD is higher for areas that receive inputs of similar strength from multiple sensory modalities, and lower when multiple inputs terminate in register with one another. Across the isocortex, TD varied by a factor of ~4. TD was positively correlated with hierarchy score, an independent measure that is based on laminar connection patterns. Furthermore, TD (an anatomical measure) was significantly related to several measures of neural activity. In particular, higher TD was associated with higher neural activity dimension, lower population sparseness, and lower lifetime sparseness of spontaneous activity, independent of an area's hierarchical position. Finally, we analyzed factors that limited TD and found that linear correlations among projections from different areas typically had little impact, while diversity of connection strengths, both between different projections onto the same area, and within projections across different parts of an area, limited TD substantially. This analysis revealed additional intricacy of cortical networks, beyond areas' sets of connections and hierarchical organization. We propose a means of approximating this organization in deep-network models.

Gender-specific Single Transcript Level Atlas of Vasopressin and its Receptor (AVPR1a) in the Mouse Brain
Vasopressin (AVP), a nonapeptide synthesized predominantly by magnocellular hypothalamic neurons, is conveyed to the posterior pituitary via the pituitary stalk, where AVP is secreted into the circulation. Known to regulate blood pressure and water homeostasis, it also modulates diverse social behaviors, such as pair-bonding, social recognition and cognition in mammals including humans. Importantly, AVP modulates social behaviors in a gender-specific manner, perhaps, due to gender differences in the distribution in the brain of AVP and its main receptor AVPR1a. There is a corpus of integrative studies for the expression of AVP and AVPR1a in various brain regions, and their functions in modulating central and peripheral actions. In order to purposefully address sexually dimorphic and novel roles of AVP on central and peripheral functions through its AVPR1a, we utilized RNAscope to map Avp and Avpr1a single transcript expression in the mouse brain. As the most comprehensive atlas of AVP and AVPR1a in the mouse brain, this compendium highlights the importance of newly identified AVP/AVPR1a neuronal nodes that may stimulate further functional studies.

GABAergic neurons from the ventral tegmental area represent and regulate force vectors
The ventral tegmental area (VTA), a midbrain region associated with motivated behaviors, consists predominantly of dopaminergic (DA) neurons and GABAergic (GABA) neurons. Previous work has suggested that VTA GABA neurons provide a reward prediction, which is used in computing a reward prediction error. In this study, using in vivo electrophysiology and continuous quantification of force exertion in head-fixed mice, we discovered distinct populations of VTA GABA neurons that exhibited precise force tuning independently of learning, reward prediction, and outcome valence. Their activity usually preceded force exertion, and selective optogenetic manipulations of these neurons systematically modulated force exertion without influencing reward prediction. Together, these findings show that VTA GABA neurons continuously regulate force vectors during motivated behavior.

In Search of Transcriptomic Correlates of Neuronal Firing-Rate Adaptation across Subtypes, Regions and Species: A Patch-seq Analysis
Can the transcriptomic profile of a neuron predict its physiological properties? Using a Patch-seq dataset of the primary visual cortex, we addressed this question by focusing on spike rate adaptation (SRA), a well-known phenomenon that depends on small conductance calcium (Ca)-dependent potassium (SK) channels. We first show that in parvalbumin-expressing (PV) and somatostatin-expressing (SST) interneurons (INs), expression levels of genes encoding the ion channels underlying action potential generation are correlated with the half-width (HW) of spikes. Surprisingly, the SK encoding gene is not correlated with the degree of SRA (dAdap). Instead, genes that encode proteins upstream from the SK current are correlated with dAdap, a finding validated by a different dataset from the mouse's primary motor cortex that includes pyramidal cells and interneurons, as well as physiological datasets from multiple regions of macaque monkeys. Finally, we construct a minimal model to reproduce observed heterogeneity across cells, with testable predictions.

Modulation of Purkinje Cell Inhibition by Stem Cell Factor
Target derived factors influence the specification, maintenance, and modulation of synaptic connectivity. The transmembrane protein, Kit Ligand, and Kit receptor tyrosine kinase are differentially expressed in connected neurons. In development and postnatal periods, these proteins maintain connectivity between cerebellar Purkinje cells (PC) that express Kit Ligand, and presynaptic Molecular Layer Interneurons (MLI) expressing Kit. In this study, it is demonstrated that Stem Cell Factor (SCF), the active extracellular domain of Kit Ligand, produces a potent potentiation of inhibition upon Purkinje Cells. The SCF enhancement of inhibition required presynaptic Kit, produced long term suppression of PC firing, and was associated with a specific potentiation of basket cells of the MLI1 subtype. It is posited that SCF exerts a postsynaptic effect involving enhanced sensitivity of somatic PC GABAA receptors. This work demonstrates that the SCF/Kit axis modulates synaptic function in adult tissue.

Grid cells anticipate the animal's future movement
Grid cells in the rodent medial entorhinal cortex preferentially fire spikes when the animal is within certain regions of space. When experimental data are averaged over time, spatial firing fields become apparent. If these firing fields represented only the current position of the animal, a grid cells firing should not depend on whether the animal is running into or out of a firing field. Yet many grid cells are sensitive to the animals direction of motion relative to the firing-field center. Such apparent egocentric "inbound-outbound tuning" could be a sign of prospective encoding of future position, but it is unclear whether grid cells code ahead in space or in time. To investigate this question, we decided to undo the inbound-outbound modulation by shifting all spikes within a given firing field by a fixed distance in space or in time. For grid-cell data recorded in mice, optimizing in space requires a forward shift of around 2.5 cm, whereas optimizing in time yielded a forward shift of about 170 ms. In either case the firing-field sizes decrease. Minimizing just the field size yields somewhat smaller shifts (roughly 1.8 cm and around 115 ms ahead). Jointly optimizing along the temporal and spatial dimension reveals a continuum of flat inbound-outbound tuning curves and a shallow minimum for field sizes, located at about 2.3 cm and 35 ms. These findings call into question a purely spatial or purely temporal interpretation of grid-cell firing fields and inbound-outbound tuning.

Multi-omic characterization of human sural nerves acrosspolyneuropathies
Diseases of peripheral nerves termed polyneuropathies (PNPs) are common, mechanistically heterogeneous, and challenging to diagnose, but have not been investigated at single cell resolution. Here, we integrated single nuclei transcriptomics of peripheral nerves from 33 human PNP patients and four controls (365,708 nuclei) with subcellular spatial transcriptomics. We identified and spatially validated novel and human-specific markers of myelinating and non-myelinating Schwann cells and unexpectedly heterogeneous perineurial fibroblasts. Nerve-associated leukocytes were diverse with an endoneurial to epineurial macrophage gradient. All PNPs shared a loss of myelinating and an increase in repair Schwann cells and lipid-associated macrophages. Transcriptional changes affected multiple cells outside of the endoneurium across PNPs, suggesting PNPs as pan-nerve diseases. Spatially, PNPs showed a perineurial hyperplasia and fibrotic dispersion and this was most pronounced in immune-mediated PNPs. We found potential of single cell transcriptomics for supporting the differential diagnosis of PNPs and for their future unbiased diagnostic classification.

Significance StatementThe first large-scale integrated single cell and spatial transcriptomic characterization of human peripheral nerves identifies novel cell markers and unexpected heterogeneity of perineurial cells, reveals polyneuropathies as pan-nerve diseases, and shows that single cell transcriptomics hold potential for unbiased nerve disease classification.

Aging and the Spectral Properties of Brain Hemodynamics
Cerebral glucose metabolism (CMRGlc) systematically decreases with advancing age. We sought to identify correlates of decreased CMRGlc in the spectral properties of fMRI signals imaged in the task-free state. We analyzed lifespan resting-state fMRI data acquired in 455 healthy adults (ages 18-87 years) and cerebral metabolic data acquired in a separate cohort of 94 healthy adults (ages 25-45 years, 65-85 years). We characterized the spectral properties of the fMRI data in terms of the relative predominance of slow vs. fast activity using the spectral slope (SS) measure. We found that the relative proportion of fast activity increases with advancing age (SS flattening) across most cortical regions. The regional distribution of spectral slope was topographically correlated with CMRGlc in young adults. Notably, whereas most older adults maintained a youthful pattern of SS topography, a distinct subset of older adults significantly diverged from the youthful pattern. This subset of older adults also diverged from the youthful pattern of CMRGlc metabolism. This divergent pattern was associated with T2-weighted signal changes in frontal lobe white matter, an independent marker of small vessel disease. These findings suggest that BOLD signal spectral slope flattening may represent a biomarker of age-associated neurometabolic pathology.

A subset of human choroid plexus epithelial cells exhibit mitochondrial eccentricity and distinct expression of the pigmentation-associated enzyme TYRP1
For decades, ultrastructural evaluation of epithelial cells in diverse organ systems has demonstrated the existence of two subtypes identified by stark differences in cytoplasmic electron density -- so-called light and dark epithelial cells. Choroid plexus (CP) epithelial cells are key regulators of CSF homeostasis and are one of many specialized epithelial linings that exhibit this bimodal phenotype. Despite longstanding acknowledgement, it has been difficult to assess the potential significance of adult human light and dark CP epithelial cells due to a lack of characterization beyond electron microscopy (EM). We present the first transcriptomic analysis of adult human CP epithelial cells and denote the existence of four epithelial subpopulations, one of which is defined by elevated expression of TYRP1 -- a melanocyte-associated tyrosine-related protein involved in cellular pigmentation and proliferation. TYRP1-high cells also downregulate genes related to cilia function (which is consistent with observations of dark cell identity in organoids) and upregulate genes associated with pathways related to cell cycling, stress, and iron regulation. Our data provide an explanation of the molecular underpinning of adult human light and dark cell identity and serve as a resource for investigations of epithelial heterogeneity in the CP and other organs where dark cells are found.

Gabapentin's Effect on Human Dorsal Root Ganglia: Donor-Specific Electrophysiological and Transcriptomic Profiles
Neuropathic pain affects approximately 10% of the adult population and is commonly treated with gabapentin (GBP), a repurposed anticonvulsant drug. Despite its widespread clinical use, GBPs efficacy varies significantly among patients, highlighting the need to better understand its functional and molecular impacts on human pain-sensing neurons. In this study, we characterized the electrophysiological and transcriptomic effects of GBP on primary sensory neurons derived from the dorsal root ganglia (DRG) of ethically consented human donors. Using patch-clamp electrophysiology, we demonstrated that GBP treatment reduced neuronal excitability, with more pronounced effects in multi-firing vs. single-firing neuronal subtypes. Notably, significant donor-specific variability was observed in electrophysiological responsiveness to GBP treatment in vitro. RNA sequencing of DRG tissue from the GBP-responsive donor revealed differences in the transcriptome-wide expression of genes associated with ion transport, synaptic transmission, inflammation, and immune response relative to non-responsive donors. Cross-transcriptomic analyses further showed that GBP treatment counteracted these altered processes, rescuing aberrant gene expression at the pathway level and for several key genes. This study provides a comprehensive electrophysiological and transcriptomic profile of the effects of GBP on human DRG neurons. These findings enhance our understanding of GBPs mechanistic actions on peripheral sensory neurons and could help optimize its clinical use for neuropathic pain management.

Tactile stimulations reduce or promote the segregation of auditory streams: psychophysics and modelling
Auditory stream segregation plays a crucial role in understanding the auditory scene. This study investigates the role of tactile stimulation in auditory stream segregation through psychophysics experiments and a computational model of audio-tactile interactions. We examine how tactile pulses, synchronized with specific tones in a sequence of interleaved high- and low-frequency tones (ABA- triplets), influence the likelihood of perceiving integrated or segregated auditory streams. Our findings reveal that tactile pulses synchronized with specific tones enhance perceptual segregation, while pulses synchronized with both tones promote integration. Based on these findings, we developed a dynamical model that captures interactions between auditory and tactile neural circuits, including recurrent excitation, mutual inhibition, adaptation, and noise. The proposed model shows excellent agreement with the experiment. Model predictions are validated through psychophysics experiments. In the model, we assume that selective tactile stimulation dynamically modulates the tonotopic organization within the auditory cortex. This modulation facilitates segregation by reinforcing specific tonotopic responses through single-tone synchronization while smoothing neural activity patterns with dual-tone alignment to promote integration. The model offers a robust computational framework for exploring cross-modal effects on stream segregation and predicts neural behaviour under varying tactile conditions. Our findings imply that cross-modal synchronization, with carefully timed tactile cues, could improve auditory perception with potential applications in auditory assistive technologies aimed at enhancing speech recognition in noisy settings.

Insights into Dentatorubral-Pallidoluysian Atrophy from a new Drosophila model of disease
Dentatorubral-pallidoluysian atrophy (DRPLA) is a neurodegenerative disorder that presents with ataxia, dementia and epilepsy. As a member of the polyglutamine family of diseases, DRPLA is caused by abnormal CAG triplet expansion beyond 48 repeats in the protein-coding region of ATROPHIN 1 (ATN1), a transcriptional co-repressor. To better understand DRPLA, we generated new Drosophila lines that express full-length, human ATN1 with a normal (Q7) or pathogenic (Q88) repeat. Expression of ATN1 is toxic, with the polyglutamine-expanded version being consistently more problematic than wild-type ATN1. Fly motility, longevity and internal structures are negatively impacted by pathogenic ATN1. RNA-seq identified altered protein quality control and immune pathways in the presence of pathogenic ATN1. Based on these data, we conducted genetic experiments that confirmed the role of protein quality control components that ameliorate or exacerbate ATN1 toxicity. Hsc70-3, a chaperone, arose as a likely suppressor of toxicity. VCP (a proteasome-related AAA ATPase), Rpn11 (a proteasome-related deubiquitinase) and select DnaJ proteins (co-chaperones) were inconsistently protective, depending on the tissues where they were expressed. Lastly, informed by RNA-seq data that exercise-related genes may also be involved in this model of DRPLA, we conducted short-term exercise, which improved overall fly motility. This new model of DRPLA will prove important to understanding this understudied disease and will help to identify therapeutic targets for it.

Comparative analysis of neural decoding algorithms for brain-machine interfaces
Accurate neural decoding of brain dynamics remains a significant and open challenge in brain-machine interfaces. While various signal processing, feature extraction, and classification algorithms have been proposed, a systematic comparison of these is lacking. Accordingly, here we conducted one of the largest comparative studies evaluating different combinations of state-of-the-art algorithms for motor neural decoding to find the optimal combination. We studied three signal processing methods (i.e., artifact subspace reconstruction, surface Laplacian filtering, and data normalization), four feature extractors (i.e., common spatial patterns, independent component analysis, short-time Fourier transform, and no feature extraction), and four machine learning classifiers (i.e., support vector machine, linear discriminant analysis, convolutional neural networks, and long short-term memory networks). Using a large-scale EEG dataset, we optimized each combination for individual subjects (i.e., resulting in 672 total experiments) and evaluated performance based on classification accuracy. We also compared the computational and memory storage requirements, which are important for real-time embedded computing. Our comparative analysis provides novel insights that help inform the design of next-generation neural decoding algorithms for brain-machine interfaces used to interact with and control robots and computers.