bioRxiv Subject Collection: Neuroscience's Journal

A simple, inexpensive battery-powered homeothermic warming pad for mice and rats
Background Anesthesia decreases core body temperature, and this seriously compromises the physiological status of an experimental animal. Hypothermia alters many aspects of neural function. When recording nervous system activity in anesthetized animals, their core temperature must be stabilized. New method This report describes an inexpensive, battery-powered, temperature-controlled warming pad for mice and rats and documents its validity and utility. The device is portable, making it convenient for researchers who conduct procedures such as surgical preparations in one location and transport the anesthetized animal to another location for experimental recordings. Results The device keeps anesthetized mice normothermic +/- 0.7 C. for over 6 hours without supplemental warmth (e.g., heat lamp), despite >15 C. differential between ambient room temperature and core body temperature. We demonstrate how the warming pad can be used for in vivo imaging of neuronal activity for a prolonged period in mice. Comparison with existing methods Commercial heating pads for small animals are expensive, somewhat bulky, and require power cords and a 120/240V source. Transporting an anesthetized animal from one location (e.g. surgical suite) to another (e.g., imaging rig) involves moving power cords. Moreover, commercial devices are not always compatible with custom stereotaxic frames, microscope stages, or holding boxes. The device described here is small, inexpensive, battery-powered, and readily adaptable to experimental set ups. Conclusion The homeothermic heating pad provides a simple method for maintaining the core temperature of anesthetized small animals. It can be constructed in under 30 minutes, the components are readily available, and the cost is less than $100. It is exceptionally useful for experiments on mice or rats.

Behaviors resulting from the activation of single olfactory receptor neuron class depends on multiple second-order neuron types
Animals rely on olfactory cues to guide critical behaviors such as foraging, mate selection, and predator avoidance. Animals discriminate between different odors because each odor binds to a distinct set of olfactory receptor neurons (ORNs). The relationship between the activated ORN class and resulting behavior is an intensely studied problem. Genetic tools in the Drosophila olfactory system make it particularly suitable for understanding this relationship. In this study, we investigate how activity in Or7a-expressing ORNs (Or7a-ORNs) which projects to the DL5 glomerulus, is transformed into aversive behavior. We find that optogenetically activating Or7a-ORNs causes an increase in locomotion speed which results in mild aversion. Surprisingly, silencing the synaptically connected second-order neuron called DL5PN increases the aversion. Silencing DL5PN has no effect on the increase in speed. The increased aversion results from the flies returning to the stimulated area less often. When DL5PN is left intact, flies return more frequently to the stimulated area. Patch-clamp recordings from PNs other than DL5PNs suggest they are activated when Or7a-ORNs are activated. These results suggest that the behavioral effect downstream of a given ORN class is mediated by multiple PN classes. This work advances our understanding of how aversion is encoded and transmitted through early sensory circuits to shape behavior.

EEG Functional Connectivity as a Marker of Evolution from Infantile Epileptic Spasms Syndrome to Lennox-Gastaut Syndrome
Timely diagnosis and effective treatment of Lennox-Gastaut Syndrome (LGS) improve prognosis and lower healthcare costs, but the transition from infantile epileptic spasms syndrome (IESS) to LGS is highly variable and insidious. Objective biomarkers are needed to monitor this progression and guide clinical decision making. We retrospectively collected longitudinal EEG data at the Children's Hospital of Orange County from fifteen children who were diagnosed with IESS and later with LGS between 2012 and 2021. EEGs were from IESS and LGS diagnoses, between the two diagnoses, and following LGS diagnosis. Functional connectivity networks were calculated using a cross-correlation-based method and assessed relative to diagnostic timepoint, treatment response, presence of clinical markers of disease, age, and amplitude of interictal spikes. Connectivity strength was high at LGS diagnosis and decreased after favorable response to treatment, but it remained stable or increased when response was unfavorable. In all subjects, connectivity strength was higher at the time of LGS diagnosis than at the preceding timepoint. Presence of clinical markers of LGS were associated with high connectivity strength, but no single marker predicted connectivity strength. Computational EEG analysis can be used to map the evolution from IESS to LGS. Changes in connectivity may enable prediction of impending LGS and treatment response monitoring, thus facilitating earlier LGS treatment and guiding medical management.

The China Brain Multi-omics Atlas Project (CBMAP)
The China Brain Multi-omics Atlas Project (CBMAP) aims to generate a comprehensive molecular reference map of over 1,000 human brains (Phase I), spanning a broad age range and multiple regions in China, to address the underrepresentation of East Asian populations in brain research. By integrating genome, epigenome, transcriptome, proteome (including multiple post-translational modifications), and metabolome data, CBMAP is set to provide a rich and invaluable resource for investigating the molecular underpinnings of aging-related brain phenotypes and neuropsychiatric disorders. Leveraging high-throughput omics data and advanced technologies, such as spatial transcriptomics, proteomics, and single-nucleus 3D chromatin structure analysis, this atlas will serve as a crucial resource for the brain science community, illuminating disease mechanisms and enhancing the utility of data from genome-wide association studies (GWAS). CBMAP is also poised to accelerate drug discovery and precision medicine for brain disorders.

Plasma pTau-217 Correlates with Brain Atrophy, Cognition, and CSF Biomarkers in a Cognitively Healthy Community Cohort
Plasma biomarkers are promising for detecting Alzheimer's disease (AD) pathology, but their role in cognitively healthy individuals remains unclear. Plasma pTau-217 has high diagnostic accuracy for clinical and prodromal AD, yet its relevance in preclinical stages is underexplored. We examined if plasma biomarkers of AD, neurodegeneration, and neuroinflammation were associated with cognition, brain structure, and their cerebrospinal fluid (CSF) counterparts in dementia-free older adults. We studied community-based, dementia-free older adults from the Brain and Cognitive Health (BACH) cohort. Neuropsychological testing assessed global cognition (MMSE), memory (Logical Memory II), visual processing (Hooper Visual Organization Test), processing speed (Trail Making Test-A), and reasoning (Similarities). Paired plasma and CSF biomarkers (pTau-217, pTau-181, GFAP, NfL, A{beta}42/40) were measured using SIMOA. MRI-derived cortical thickness was used as a neurodegeneration marker. Multivariable linear regression assessed associations between log10-transformed plasma biomarker levels, cognition (adjusted for age, sex, education, hypertension, hyperlipidemia, diabetes), and cortical thickness (adjusted for age, sex, education, and intracranial volume). Pearson's correlations and Bland-Altman plots evaluated plasma-CSF agreement. There were 147 dementia-free participants (mean age{+/-}SD: 66.7{+/-}7.7 years; 56 % women). Higher plasma pTau-217 levels associated with lower global cognition scores ({beta} -0.80, 95% C.I. -1.56, -0.03, p=0.041) and abstract reasoning ({beta} -0.86, 95% confidence interval [C.I.] -1.62, -0.09, p=0.028). Greater plasma pTau-217 also associated with lower global cortical thickness ({beta}eta [{beta}] -0.21, 95% confidence interval [C.I]. -0.37, -0.06, per log unit change; p=0.006). No associations were found between the plasma biomarkers and processing speed or visual processing (p>0.05 for all). Among 47 participants with paired plasma-CSF biomarkers, plasma pTau-217 showed the strongest correlation with its CSF counterpart (R=0.76, p<0.0001), outperforming pTau-181 (R=0.61, p<0.0001), GFAP (R=0.66, p<0.0001), NfL (R=0.56, p<0.0001), and A{beta}42/40 (R=0.53, p=0.0001). In conclusion, plasma pTau-217 levels were associated with both cognition and cortical thickness in dementia-free older adults. All plasma biomarkers correlated significantly with their CSF counterpart. These findings reinforce the utility of plasma biomarkers, particularly pTau-217, as indicators of neurodegenerative processes, even in asymptomatic individuals.

Mechanical Artifacts During Transcranial Focused Ultrasound Mimic Biologically Evoked Responses
Low-intensity transcranial focused ultrasound stimulation (tFUS) has emerged as a promising modality for non-invasive neuromodulation, offering deep brain penetration and high spatial precision. However, the electrophysiological effects of tFUS remain poorly understood, in part due to challenges distinguishing genuine neural responses from mechanical artifacts. In this study, we investigated the electrophysiological signatures captured during tFUS of the anesthetized rat hippocampus using silicon microelectrodes. We observed a strong, stereotyped local field potential (LFP) response that was time-locked to the onset and offset of sonication and resembled sensory-evoked potentials. Critically, the same waveform was observed in euthanized animals, confirming a non-biological, artifact-driven origin. The artifact scaled with acoustic intensity and was most pronounced under continuous-wave sonication. These findings suggest that electrode movement induced by ultrasound can generate artifactual LFP signals that closely mimic authentic neural responses. Our results underscore the need for caution when interpreting in situ electrophysiological recordings during tFUS and advocate for alternative, artifact-resistant readouts such as fiber photometry to unambiguously detect neuromodulatory effects.

Daily light exposure habits of youth with migraine: A prospective pilot study
Background: Eighty percent of youth with migraine report photophobia. It is unknown if photophobia leads to light avoidant behavior, and if such behaviors worsen light sensitivity and disrupt sleep. Recently developed wearable, continuous light loggers allow us to address these open questions. We conducted a pilot study to determine the feasibility of measuring light exposure using wearable light loggers in youth with migraine. Methods: Youth 10 to 21 years old with a headache-specialist confirmed ICHD-3 diagnosis of migraine were recruited from CHOP headache clinics. Each participant recorded 7 consecutive days of light logging data from the ActLumus device worn as a pendant around the neck paired with a text-based daily migraine symptom diary during a typical school week between November and March 2024. Validated questionnaires were used to capture headache and migraine frequency, headache-related disability, visual sensitivity, fear-of-pain, and sleep disturbance and impairment. Percent time spent within recommended light exposure levels was calculated for the day, 3 hours prior to bedtime, and night. Power analysis was calculated to determine sample size needed for group comparison of baseline characteristics across light intensity and light timing metrics to aid in the design of larger studies. Results: Twenty youth with a median age 17 years [IQR 16, 19], 70% of whom were female completed 7 days of continuous light logger recording and daily headache diary. Data completion rates were high with 136/140 (97.1%) useable days of light logger data, and 100% compliance on the daily headache diary. Participant feedback on the study was positive; 85% would recommend the study to others. On average, participants received recommended light exposure during only 14.5% +/- SD 7.0 of daylight hours. By contrast, participants were more consistently below the recommended maximum light levels 3 hours prior to bed (77.5% +/- 21.6 of the time), and at night (99.1% +/- 2.9 of the time). Youth with chronic migraine (i.e., at least 15 headache days and 8 migraine days per month) had daily light exposure patterns that were phase shifted 60 minutes later as compared to participants with non-chronic migraine. Power analyses suggest that future tests for differences in light exposure between migraine-characteristic groups (e.g., differing by headache frequency, severity, or disability) will require sample sizes on the order of 50 to 150 to reach 80% power with an alpha of 0.05. Conclusion: Measuring daily light exposure is feasible in pediatric populations with photophobia and reveals intriguing trends in youth with migraine that warrant further study.

Network Neuroscience of Human Multitasking: Local Features Matter
The neural basis of multitasking costs is subject to continuing debate. Cognitive theories assume that overlap of task representations may lead to between-task crosstalk in concurrent task processing and thus requires cognitive control. Recent research suggests that modality-based crosstalk contributes to multitasking costs, involving central overlap of modality-specific representations. Consistently increased costs for specific modality pairings (visual-vocal and auditory-manual vs. visual-manual and auditory-vocal) were demonstrated (modality-compatibility effect), which were recently linked to representational overlap in the auditory cortex. However, it remains unclear whether modality-based crosstalk emerges from overlapping patterns of global brain connectivity and whether resolving it requires additional involvement of cognitive control as reflected in the fronto-parietal control network. This preregistered functional imaging study investigates these questions in 64 healthy, young human adults. Specifically, we focus on the modality-compatibility effect in multitasking by employing functional connectivity (FC) analysis. First, we tested the FC similarity between the single-task networks. Second, we compared the strength of the control network in whole-brain FC between dual tasks. We found no evidence for different FC similarities of single-task networks between modality pairings and no additional involvement of the control network during dual tasks. However, post-hoc connectivity analysis revealed a brain-behavior correlation for the modality-compatibility effect in dual tasks. This effect was locally restricted to FC between lateral frontal and sensory auditory regions, providing evidence for the modality-based crosstalk theory. More generally, the findings suggest that robust behavioral differences in multitasking are not necessarily related to global functional connectivity differences but to local connectivity changes.

An open, fully-processed data resource for studying mood and sleep variability in the developing brain
Brain development during adolescence and early adulthood coincides with shifts in emotion regulation and sleep. Despite this, few existing datasets simultaneously characterize affective dynamics, sleep variation, and multimodal measures of brain development. Here, we describe the study protocol and initial release (n = 10) of an open data resource of neuroimaging paired with densely sampled behavioral measures nd in adolescents and young adults. All participants complete multi-echo functional MRI, compressed-sensing diffusion MRI, and advanced arterial spin-labeled MRI. Behavioral measures include ecological momentary assessment, actigraphy, extensive cognitive assessments, and detailed clinical phenotyping focused on emotion regulation. Raw and processed data are openly available without a data use agreement and will be regularly updated as accrual continues. Together, this resource will accelerate research on the links between mood, sleep, and brain development.

Centroparietal signals track multiple rounds of evidence accumulation in an intermittent evidence task
Making decisions often requires the integration of multiple pieces of information. An extensive body of research has investigated the neural mechanisms underpinning evidence accumulation in tasks where information is continuously present, but less is known about how this neural architecture supports evidence accumulation if the flow of information is paused for a variable amount of time. In two electroencephalography (EEG) experiments (N = 22, N = 21), participants made perceptual decisions about two pulses of motion evidence separated by gaps of varying duration. Our goal was to investigate 1) whether and how people integrate the two pulses across various delay durations, and 2) how a prominent EEG marker of evidence accumulation, the centroparietal positivity (CPP), reflects decisions in the context of our intermittent-pulses task. Our results are consistent with a bounded evidence accumulation process where the second pulse further informs the decision only if the bound is not reached by the first pulse. We show that whereas motor preparation signals reflected in motor beta lateralisation (MBL) maintain the cumulative evidence level reached from pulse 1 through the gap, the CPP does not sustain but rather exhibits transient deflections following each pulse, encoding momentary belief updates.

Overexpression of Meis factors in late-stage retinal progenitors yields complex effects on temporal patterning and neurogenesis.
The vertebrate retina serves as a model for studying neurogenesis and cell fate specification, with retinal progenitor cells following a tightly regulated temporal sequence to generate distinct cell types. Meis1 and Meis2 are transcription factors implicated in early retinal development, but their role in late-stage RPCs remains poorly understood. Here, we investigate whether Meis1 and Meis2 overexpression in postnatal mouse RPCs can alter temporal identity and induce early-born cell types. Using electroporation and single-cell RNA sequencing, we find that while these factors modestly upregulate early-stage gene regulatory network components, they do not repress late-stage transcription factors or induce early-born retinal cells. Meis1 overexpression reduces proliferation and inhibits neurogenesis, whereas Meis2 overexpression accelerates neurogenic progression without altering fate commitment. Our findings suggest that overexpression of Meis1 and Meis2 modulate largely non-overlapping aspects of temporal identity and neurogenic competence but are insufficient to fully reprogram late-stage progenitors. These results have implications for regenerative strategies aimed at reprogramming retinal cells for therapeutic purposes.

Self-Organizing Neural Networks in Organoids Reveal Principles of Forebrain Circuit Assembly
The mouse cortex is a canonical model for studying how functional neural networks emerge, yet it remains unclear which topological features arise from intrinsic cellular organization versus external regional cues. Mouse forebrain organoids provide a powerful system to investigate these intrinsic mechanisms. We generated dorsal (DF) and ventral (VF) forebrain organoids from mouse pluripotent stem cells and tracked their development using longitudinal electrophysiology. DF organoids showed progressively stronger network-wide correlations, while VF organoids developed more refined activity patterns, enhanced small-world topology, and increased modular organization. These differences emerged without extrinsic inputs and may be driven by the increased generation of Pvalb+ interneurons in VF organoids. Our findings demonstrate how variations in cellular composition influence the self-organization of neural circuits, establishing mouse forebrain organoids as a tractable platform to study how neuronal populations shape cortical network architecture.

Targeted plasma proteomics uncover novel proteins associated with KIF5A-linked SPG10 and ALS spectrum disorders
KIF5A (Kinesin family member 5A) is a motor protein that functions as a key component of the axonal transport machinery. Variants in KIF5A are linked to several neurodegenerative diseases, mainly spastic paraplegia type 10 (SPG10), Charcot-Marie-Tooth disease type 2 (CMT2), and amyotrophic lateral sclerosis (ALS). These diseases share motor neuron involvement but vary significantly in clinical presentation, severity, and progression. KIF5A variants are mainly categorized into N-terminal variants associated with SPG10/CMT2 and C-terminal variants linked to ALS. This study utilized a novel multiplex NULISA targeted platform to analyze plasma proteome from KIF5A-linked SPG10, ALS patients and compared to healthy controls. Our results revealed distinct proteomic signatures, with significant alterations in proteins related to synaptic function, and inflammation. Notably, neurofilament light polypeptide, a biomarker for neurodegenerative diseases, was elevated in KIF5A ALS but not in SPG10 patients. Moreover, these findings can now be taken forward to gain mechanistic understanding of axonopathies linking to N- vs C-terminal KIF5A variants affecting both central and peripheral nervous systems.

MRI-Derived Markers of Acute and Chronic Inflammatory Processes in the VTA Associated with Depression
Background: Depression is a leading cause of disability worldwide, with inflammation increasingly recognized as a contributing factor. Inflammatory processes can disrupt the brain's reward circuitry, particularly the ventral tegmental area (VTA), which is central to dopamine-mediated motivation and reward. This study investigates whether MRI-derived markers sensitive to neuroinflammation and microstructure in the VTA are associated with depression diagnosis and symptom severity. Methods: We analyzed diffusion weighted imaging and quantitative susceptibility mapping data from 32,495 UK Biobank participants, including 3,807 individuals with ICD-10 diagnosed depression. Metrics sensitive to neuroinflammation (free water [FW], isotropic volume fraction [ISOVF], magnetic susceptibility) and microstructure (intracellular volume fraction [ICVF], orientation dispersion index [ODI] volume) were extracted from the VTA. Group differences between the major depression group and BMI, sex, and age-matched healthy controls were assessed using ANOVAs and linear regression was used to predict acute symptom severity based on Recent Depressive Symptoms scores. Results: Participants with depression diagnosis had significantly higher FW ({rho} < 0.001) and ISOVF ({rho} = 0.001) compared to HCs, indicating increased extracellular processes such as inflammation in the VTA. Lower ISOVF ({beta} = -0.46, {rho} = 0.017) and higher ICVF ({beta} = 0.34, {rho} = 0.007) and ODI ({beta} = 0.49, {rho} = 0.004) were associated with higher depression severity, independent of depressive diagnosis history. Conclusions: Our findings reveal distinct patterns of VTA microstructural changes associated with depression history versus acute depressive symptom severity, suggesting different underlying pathophysiological mechanisms. Distinct patterns of neuroinflammation may differentiate acute from chronic depression, informing targeted interventions.

Targeting Lysosomal Dysfunction to Alleviate Plaque Deposition in an Alzheimer Disease Model
Alzheimer disease (AD) is characterized by aberrant amyloid precursor protein (APP) processing and lysosomal dysfunction. This study identifies two members of the lysosomal multi-enzyme complex (LMC), neuraminidase 1 (Neu1) and protective protein/cathepsin A (PPCA), as a critical regulators of APP metabolism. Neu1 deficiency in human AD brains and 5xFAD/Neu1-/- mice leads to sialic acid retention on APP and its secretases, enhancing amyloidogenic cleavage and A{beta}42 production. Additionally, Neu1 deficiency increases lysosomal exocytosis, contributing to extracellular A{beta} release and neuroinflammation. Conversely, overexpression of PPCA in neurons or co-expression of PPCA and NEU1 normalizes sialylation patterns, reduces secretase activity, and mitigates plaque burden. These findings reveal a novel bidirectional dependency between Neu1 and PPCA, underscoring their cooperative role in maintaining lysosomal homeostasis. Additionally, AAV-mediated co-expression of NEU1 and PPCA in 5XFAD brains demonstrates therapeutic potential by reducing amyloid pathology. These findings position lysosomal dysfunction and the Neu1-PPCA axis as promising targets for therapeutic intervention in AD.

Single-cell multiomics identifies both shared and unique features of immune dysfunction in the colon, plasma and stool from individuals diagnosed with Parkinson's disease or inflammatory bowel disease
Parkinson's disease (PD) is the fastest growing neurodegenerative disease in the world. Gastrointestinal (GI) dysfunction can occur decades before motor impairments and in up to 80% of individuals living with PD. We investigated peripheral relationships that may underlie mechanisms along the gut-blood axis that contribute to PD pathogenesis. Single-cell multiomic spatial molecular imaging (SMI) of colonic tissue localized inflammatory injury within epithelial cells that appear to be associated with iron mishandling in both inflammatory bowel disease (IBD) and PD biosamples. We found that both the single-cell SMI of RNA and protein revealed parallel cross-modal dysregulation in the gut epithelium, in both IBD and PD biosamples. These data are accompanied by plasma (PD) and stool (IBD) protein depletion of CCL22. Our findings suggest iron mishandling along the gut barrier likely contributes to systemic inflammation, which may be the catalyst that primes circulating immune cells to body-first PD pathogenesis.

Molecular and Spatial Organization of the Primary Olfactory System and its Responses to Social Odors
The detection of olfactory cues is essential to signal food, predators, and social encounters. To determine how the sensory detection of physiologically relevant odors is systematically mapped into the mouse primary olfactory system, we used Multiplexed Error Robust Fluorescent In Situ Hybridization (MERFISH) to construct a molecular atlas of odorant receptor (OR) expression in the main olfactory epithelium (MOE) and olfactory bulb (OB). We comprehensively quantified the expression of the mouse OR repertoire and uncovered stereotypical gradients of sensory neuron distribution in the MOE along two, central-to-peripheral and basal-to-apical, axes. Projections of sensory neurons mirror MOE gradients along the dorsal-ventral and anterior-posterior axes of the OB, respectively. Integration with sequencing data revealed candidate signaling molecules underlying this spatial organization. Co-imaging OR and activity marker expression identified distinct spatial domains of sensory responses in the MOE and OB, providing a topographical basis for olfactory responses to ethologically relevant odors.