bioRxiv Subject Collection: Neuroscience's Journal

DANCE: An open-source analysis pipeline and low-cost hardware to quantify aggression and courtship in Drosophila
Quantifying animal behaviors is pivotal for identifying the underlying neuronal and genetic mechanisms. Computational approaches have enabled automated analysis of complex behaviors such as aggression and courtship in Drosophila. However, existing approaches rely on rigid, rule-based algorithms and expensive hardware, limiting sensitivity to behavioral variations and accessibility. Here, we describe the DANCE (Drosophila Aggression and Courtship Evaluator), a low-cost, open-source platform combining machine learning-based classifiers and inexpensive hardware to quantify aggression and courtship. DANCE consists of six novel behavioral classifiers trained using a supervised machine learning algorithm. DANCE classifiers outperform existing rule-based algorithms by capturing dynamic behavioral variations. DANCE hardware is constructed using repurposed medicine blister packs and acrylic sheets, with recordings performed using smartphones, making it affordable and accessible. Benchmarking demonstrated that DANCE hardware performs comparably to sophisticated, high-cost setups. We validated DANCE in diverse contexts, including social isolation versus enrichment that modulate aggression and courtship, RNAi-mediated down-regulation of neuropeptide Dsk, and optogenetic silencing of dopaminergic neurons which promoted aggression. DANCE provides a cost-effective and portable solution for studying Drosophila behaviors in resource-limited settings or closer to natural habitats. Its accessibility and robust performance democratizes behavioral neuroscience, enabling rapid screening of genes and neuronal circuits underlying complex social behaviors.

Linking neural population formatting to function
Animals capable of complex behaviors tend to have more distinct brain areas than simpler organisms, and artificial networks that perform many tasks tend to self-organize into modules (1-3). This suggests that different brain areas serve distinct functions supporting complex behavior. However, a common observation is that essentially anything that an animal senses, knows, or does can be decoded from neural activity in any brain area (4-6). If everything is everywhere, why have distinct areas? Here we show that the function of a brain area is more related to how different types of information are combined (formatted) in neural representations than merely whether that information is present. We compared two brain areas: the middle temporal area (MT), which is important for visual motion perception (7, 8), and the dorsolateral prefrontal cortex (dlPFC), which is linked to decision-making and reward expectation (9,10)). When monkeys based decisions on a combination of motion and reward information, both types of information were present in both areas. However, they were formatted differently: in MT, they were encoded separably, while in dlPFC, they were represented jointly in ways that reflected the monkeys' decision-making. A recurrent neural network (RNN) model that mirrored the information formatting in MT and dlPFC predicted that manipulating activity in these areas would differently affect decision-making. Consistent with model predictions, electrically stimulating MT biased choices midway between the visual motion stimulus and the preferred direction of the stimulated units (11), while stimulating dlPFC produced 'winner-take-all' decisions that sometimes reflected the visual motion stimulus and sometimes reflected the preference of the stimulated units, but never in between. These results are consistent with the tantalizing possibility that a modular structure enables complex behavior by flexibly reformatting information to accomplish behavioral goals.

TSPO-PET Reveals Higher Inflammation in White Matter Disrupted by Paramagnetic Rim Lesions in Multiple Sclerosis
Objective: To explore whether the inflammatory activity is higher in white matter (WM) tracts disrupted by paramagnetic rim lesions (PRLs) and if inflammation in PRL-disrupted WM tracts is associated with disability in people with multiple sclerosis (MS). Methods: Forty-four MS patients and 16 healthy controls were included. 18 kDa-translocator protein positron emission tomography (TSPO-PET) with the 11C-PK11195 radioligand was used to measure the neuroinflammatory activity. The Network Modification Tool was used to identify WM tracts disrupted by PRLs and non-PRLs that were delineated on MRI. The Expanded Disability Status Scale was used to measure disability. Results: MS patients had higher inflammatory activity in whole brain WM compared to healthy controls (p=0.001). Compared to patients without PRLs, patients with PRLs exhibited higher levels of inflammatory activity in the WM tracts disrupted by any type of lesions (p=0.02) or PRLs (p=0.004). In patients with at least one PRL, inflammatory activity was higher in WM tracts highly disrupted by PRLs compared to WM tracts highly disrupted by non-PRLs (p=0.009). Elevated inflammatory activity in highly disrupted WM tracts was associated with increased disability in patients with PRL (p=0.03), but not in patients without PRL (p=0.2). Interpretation: This study suggests that patients with PRLs may exhibit more diffuse WM inflammation in addition to higher inflammation along WM tracts disrupted by PRLs compared to non-PRLs, which could contribute to larger lesion volumes and faster disability progression. Imaging PRLs may serve to identify patients with both focal and diffuse inflammation, guiding therapeutic interventions aimed at reducing inflammation and preventing progressive disability in MS.

Neuronal LAG3 facilitates pathogenic α-synuclein neuron-to-neuron propagation
Lymphocyte activation gene 3 (LAG3) is a key receptor involved in the propagation of pathological proteins in Parkinson disease (PD). This study investigates the role of neuronal LAG3 in mediating the binding, uptake, and propagation of alpha-synuclein (aSyn) preformed fibrils (PFFs). Using neuronal LAG3 conditional knockout mice and human induced pluripotent stem cells-derived dopaminergic (DA) neurons, we demonstrate that LAG3 expression is critical for pathogenic aSyn propagation. Our results show that the absence of neuronal LAG3 significantly reduces aSyn pathology, alleviates motor dysfunction, and inhibits neurodegeneration in vivo. Electrophysiological recordings revealed that aSyn PFFs induce pronounced neuronal hyperactivity in wild-type (WT) neurons, increasing firing rates in cell-attached and whole-cell configurations, and reducing miniature excitatory postsynaptic currents. In contrast, neurons lacking LAG3 resisted these electrophysiological effects. Moreover, treatment with an anti-human LAG3 antibody in human DA neurons inhibited aSyn PFFs binding and uptake, preventing pathology propagation. These findings confirm the essential function of neuronal LAG3 in mediating aSyn propagation and associated disruptions, identifying LAG3 as a potential therapeutic target for PD and related alpha-synucleinopathies.

Convergent state-control of endogenous opioid analgesia
Pain is a dynamic and nonlinear experience shaped by injury and contextual factors, including expectations of future pain or relief. While mu opioid receptors are central to the analgesic effects of opioid drugs, the endogenous opioid neurocircuitry underlying pain and placebo analgesia remains poorly understood. The ventrolateral column of the posterior periaqueductal gray is a critical hub for nociception and endogenous analgesia mediated by opioid signaling. However, significant gaps remain in understanding the cell-type identities, the sub-second neural dynamics involved in pain modulation, the role of endogenous peptide neuromodulators, and the contextual factors influencing these processes. Using spatial mapping with single-nuclei RNA sequencing of pain-active neurons projecting to distinct long-range brain targets, alongside cell type-specific and activity-dependent genetic tools for in vivo optical recordings and modulation of neural activity and opioid peptide release, we identified a functional dichotomy in the ventrolateral periaqueductal gray. Neurons expressing mu opioid receptors encode active nociceptive states, whereas enkephalin-releasing neurons drive pain relief during recovery from injury, in response to learned fear predictions, and during placebo analgesia. Finally, by leveraging the functional effects of placebo analgesia, we used direct optogenetic activation of vlPAG enkephalin neurons to drive opioid peptide release, resulting in a robust reduction in pain. These findings show that diverse need states converge on a shared midbrain circuit that releases endogenous opioids with high spatiotemporal precision to suppress nociceptive activity and promote analgesia.

Circadian Rhythms are Disrupted in Patients and Preclinical Models of Machado-Joseph Disease
Machado-Joseph disease (MJD) is caused by an abnormal CAG repeat expansion in the ATXN3 gene, leading to the expression of a mutant ataxin-3 (mutATXN3) protein. MJD patients exhibit a wide range of clinical symptoms, including motor incoordination. Emerging evidence highlights circadian rhythm disruptions as early indicators and potential risk factors for the progression of neurodegenerative conditions. Circadian rhythms are regulated by internal clocks, with the suprachiasmatic nucleus (SCN) acting as the master pacemaker to synchronize timing across the behavioural and physiological functions. While sleep disturbances have been observed in MJD, the role of clock regulation in its pathophysiology remains largely unexplored in spinocerebellar ataxias. This study aimed to investigate circadian rhythms, characterize associated disruptions, and uncover the mechanisms underlying clock dysregulation in patients and preclinical models of MJD. Circadian activity in MJD patients was assessed over two weeks using actigraphy, while in a YAC-MJD transgenic mouse model, circadian rhythms were examined through: (a) wheel-running experiments; (b) telemetry-based monitoring of core body temperature; (c) immunohistochemical analysis of the neuropeptides arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) in the SCN and paraventricular nucleus (PVN); and (d) RT-qPCR evaluation of clock gene expression in the cerebellum. The impact of mutATXN3 on clock mechanisms was further investigated using Bmal1/Per2-luciferase reporters. MJD patients exhibited a progressive decline in robustness of behavioural rhythms, demonstrated by negative correlations between the circadian function index, rest-activity fragmentation, and sleep efficiency with MJD clinical scales. YAC-MJD mice exhibited reduced activity levels, increased behavioural fragmentation, and required three additional days to re-entrain after a jet lag protocol, compared to controls. Disrupted core body temperature rhythms were observed, including a phase advance and elevated temperature at the onset of the active period. Furthermore, transgenic mice showed reduced levels of VIP and AVP in the SCN and PVN, and decreased clock gene expression in the cerebellum. Lastly, we found new mechanistic evidence that WT ATXN3 activates the promoters of Bmal1 and Per2, whereas mutATXN3 loses the capacity to drive Per2 upon polyglutamine expansion. Overall, our findings indicate that central clock dysfunction in MJD is associated with impaired clock gene expression and disruptions in activity and temperature rhythms. This study provides the first robust evidence of circadian rhythm dysregulation and underlying mechanisms in MJD, paving the way for the identification of new biomarkers and the development of novel circadian-based interventions to tackle MJD and possibly other spinocerebellar ataxias.

Transcriptomic analysis of repeat expansion-ataxias uncovers distinct non-neuronal cell type-specific signatures of disease across the human brain
Hereditary ataxias are a heterogeneous group of neurogenetic conditions characterised by the clinical syndrome of progressive loss of coordination from neurodegeneration of the cerebellum. A commonality across the most prevalent ataxias is the underlying disease mechanism secondary to expansions of short tandem DNA repeats. There is currently an incomplete understanding of the pathogenic mechanisms of these repeat expansion disorders, a core feature of which revolves around RNA-dysregulation. In this study, we used both bulk and single nuclear RNA-sequencing to study post-mortem brain tissue of human donors with a range of repeat-expansion ataxias to reveal further mechanistic insights. We compared post-mortem paired cerebellar and frontal cortex tissue bulk RNA-sequencing data from 23 ataxia patients and 22 sex-, age-matched controls from two brain banks (spinocerebellar ataxia (SCA)1, SCA2, SCA6, SCA7, SCA17, Friedreich's ataxia (FRDA), and 7 cases with unknown molecular diagnoses). We analysed bulk RNA-sequencing data for transcript usage, differential and cell-type-specific expression to transcriptomically profile these diseases. We also generated single nuclear RNA-sequencing data of the cerebellum from donors with SCA1, SCA2, SCA6 and FRDA to decipher changes in cell type proportions in the disease state. Using this approach, we found that: (i) despite the commonalities in the genetics of ataxia, there were components of their transcriptional signatures which were distinct; (ii) there were extensive transcriptional changes evident not only in the cerebellum but also the frontal cortex in ataxia cases; (iii) activation of immune and inflammatory pathways, as well as involvement of non-neuronal cell types was a feature of all ataxias to a lesser or greater extent. This study provides a novel resource to understand the mechanisms of disease in ataxia. Furthermore, taken together, these results highlight immune pathways and the role of non-neuronal cell types as early and potentially important therapeutic targets. These findings provide a map of transcriptomic changes in ataxia to further understanding of the underlying pathogenesis.

Human iPSC-based coculture model reveals neuroinflammatory crosstalk between microglia and astrocytes
Background: Microglia and astrocytes have been implicated as central mediators of neuroinflammatory processes in several neurodegenerative diseases. However, their intricate crosstalk and contributions to pathogenesis remain elusive, highlighting the need for innovative in vitro approaches for investigating glial interactions in neuroinflammation. The aim of this study was to develop advanced human-based glial coculture models to explore the inflammatory roles and interactions of microglia and astrocytes in vitro. Methods: We utilized human induced pluripotent stem cell (iPSC)-derived microglia and astrocytes cultured both in conventional culture dishes and in a compartmentalized microfluidic chip coculture platform. This novel platform features separate compartments for both cell types, enabling the creation of fluidically isolated microenvironments with spontaneous migration of microglia toward astrocytes through interconnecting microtunnels. To induce inflammatory activation, glial cultures were stimulated with lipopolysaccharide (LPS), a combination of tumor necrosis factor- (TNF-) and interleukin-1{beta} (IL-1{beta}), or interferon-{gamma} (IFN-{gamma}) for 24 hours. The glial activation and crosstalk were analyzed with immunocytochemistry, the secretion of inflammatory factors from the culture media was measured, and microglial migration was quantified. Results: Microglia-astrocyte cocultures were successfully generated in both conventional cultures and the microfluidic chip platform. Inflammatory stimulation with LPS and TNF-/IL-1{beta} elicited cell type-specific responses in microglia and astrocytes, respectively. Notably, the levels of secreted inflammatory mediators were altered under coculture conditions, revealing significant glial crosstalk. Utilization of our microfluidic coculture platform facilitated the study of microglial migration and glial activation within distinct inflammatory microenvironments. Microglia migrated efficiently toward the astrocyte compartment, and the chemoattractant adenosine diphosphate (ADP) notably increased microglial migration within this platform. Furthermore, inflammatory stimulation of the microfluidic chip cocultures successfully recapitulated glial crosstalk, revealing unique responses. This crosstalk was associated with elevated levels of complement component C3 in the cocultures, emphasizing the intricate interplay between microglia and astrocytes under inflammatory conditions. Conclusions: Our results depict an elaborate molecular crosstalk between inflammatory microglia and astrocytes, providing evidence of how glial cells orchestrate responses during neuroinflammation. Importantly, we demonstrate that the microfluidic coculture platform developed in this study for microglia and astrocytes provides a more functional and enhanced setup for investigating inflammatory glial interactions in vitro.

Valence-dependent sensory-rhythmic neural entrainment modulates cortico-subcortical dynamics, attention, and memory
Recent studies have shown that sensory rhythmic stimulation can enhance executive functions by synchronizing, entraining, oscillations within higher-order cortical networks. However, whether this entrainment extends to subcortical structures and shapes human behavior remains unclear. In a first experiment, we used intracranial EEG recordings in epileptic patients during a visual search task. While neutral stimulation induced moderate entrainment, we demonstrated that 5 Hz negative-valence stimulation can significantly entrain theta oscillations in a widespread task-related cortico-subcortical network, with increased synchrony in the ventral visual stream, the hippocampus, and the dorsolateral prefrontal cortex. In a second behavioral experiment in healthy individuals, visual search performance improved under both stimulation conditions, but memory for target images, assessed through an additional recognition task, was significantly enhanced after negative stimulation. These findings unravel the role of valence in modulating subcortical brain activity and behaviors through rhythmic sensory stimulation and pave the way for its further application in clinical intervention.

Emergence of a contrast-invariant representation of naturalistic texture in macaque visual cortex
Sensory stimuli vary across a variety of dimensions, like contrast, orientation, or texture. The brain must rely on population representations to disentangle changes in one dimension from changes in another. To understand how the visual system might extract separable stimulus representations, we recorded multiunit neuronal responses to texture images varying along two dimensions: contrast, a property represented as early as the retina, and naturalistic statistical structure, a property that modulates neuronal responses in V2 and V4, but not in V1. We measured how sites in these 3 cortical areas responded to variation in both dimensions. Contrast modulated responses in all areas. In V2 and V4, the presence of naturalistic structure both modulated responses and increased contrast sensitivity. Tuning for naturalistic structure was strongest in V4; tuning in both dimensions was most heterogeneous in V4. We measured how well populations in each area could support the linear readout of both dimensions. Populations in V2 and V4 could support the linear readout of naturalistic structure, but only in V4 did we find evidence for a robust representation that was contrast-invariant.

Antidepressants promote developmental-like plasticity through remodeling of extracellular matrix
Selective serotonin reuptake inhibitors like Fluoxetine (Flx) are widely used to treat mood and anxiety disorders. Despite decades of use, the molecular logic by which they modulate stress-related behaviors remain poorly understood. Here we show that Flx reactivates a developmental-like plasticity program in the dentate gyrus (DG) by remodeling the extracellular matrix (ECM). Single-nucleus RNA sequencing of the hippocampus post-Flx revealed robust transcriptomic reprogramming in the DG, with mature granule cells adopting a juvenile-like transcriptional profile, including upregulation of the developmental gene Sox11. This was accompanied by increased BDNF signaling and enhanced structural remodeling in the mossy fiber pathway. Flx remodeled perisynaptic nets, a novel ECM structures in the DG, and targeted ECM degradation around granule cells reactivated SOX11. Flx treatment mitigated stress-induced fear generalization, and this was phenocopied by direct degradation of ECM in the DG. These findings identify ECM remodeling as a molecular substrate for Flx's effects, linking induction of a developmental-like plasticity program and rejuvenation of mature granule cells to improvements in stress-induced overgeneralization.

Short-Term Arcade-Action Video Gaming Enhances Working Memory Precision
Objective: Action video games are increasingly recognized for their potential to enhance cognitive capacities, including working memory (WM). However, most studies require extensive training or prior gaming experience. We examined whether a single, short-term session of Snail Mail, an arcade-action video game, could boost WM precision compared to a passive control condition. Method: Thirty six healthy adults (18 34 years) were randomly assigned to either play Snail Mail (Intervention) or watch episodes of Shaun the Sheep (Control) for 30 minutes. Two participants were excluded for unstable performance, yielding a final sample of 34. Participants completed a face emotion recognition memory task before and after the intervention, wherein the absolute error served as the primary metric of WM precision. A mixture model further characterized memory precision, and random guessing. Results: The Intervention group showed a significant reduction in absolute error from pre test to posttest, whereas the Control group exhibited a negative effect size, indicating worsened posttest performance. Mixture model results revealed a significant increase in precision for the Intervention group. Conclusion: These findings indicate that a 30 minute arcade-action game session can meaningfully enhance WM precision, highlighting the feasibility of brief, targeted gaming interventions. Future research should explore longer-term outcomes, generalizability to other cognitive tasks, and optimal intervention protocols for sustained benefits

Early Noise Exposure and Changes in Medial Olivocochlear Strength Alters Auditory Pathway Development
Periodic spontaneous activity is a key feature of developing sensory systems, essential for the formation and refinement of neural circuits. Before the onset of hearing in altricial mammals, cochlear inner hair cells exhibit spontaneous electrical activity that activates primary afferents to propagate into the central nervous system. This activity, modulated by the medial olivocochlear efferent feedback via 910 nicotinic cholinergic receptors present in inner hair cells, is crucial for auditory system maturation. In this study, we examined the impact of sound exposure at levels below those causing hair cell damage during the critical developmental period by using genetically modified mice - 9 knockout and 9 knock-in models - with either absent or enhanced cholinergic activity. We further assessed how varying levels of medial olivocochlear feedback interact with early-age noise exposure to affect auditory development. Our findings reveal that both increased and absent olivocochlear activity result in altered auditory sensitivity at the onset of hearing and affect ribbon synapse number and morphology. Additionally, early exposure to loud noise caused long lasting changes in the inner ear of wild-type and 9 knockout mice, underscoring the heightened vulnerability during early development. In contrast, mice with enhanced cholinergic activity were protected from these changes. Overall, this work highlights the critical role of medial olivocochlear modulation in proper auditory system development and indicates that early noise exposure can interfere with normal cochlear maturation, resulting in pronounced long-term effects.

Altered brain-ventricle coupling modes over Alzheimer's disease progression detected with fMRI
The origins of resting-state functional MRI (rsfMRI) signal fluctuations remain debated. Recent evidence shows coupling between global cortical rsfMRI signals and cerebrospinal fluid inflow in the fourth ventricle, increasing during sleep and decreasing with Alzheimer's disease (AD) progression, potentially reflecting brain clearance mechanisms. However, the existence of more complex brain-ventricle coupling modes and their relationship to cognitive decline remains unexplored. Analyzing 599 minimally-preprocessed rsfMRI scans from 163 elderly participants across the AD spectrum, we identified distinct brain-ventricle coupling modes that differentiate across groups and correlate with cognitive scores. Beyond the known anti-phase coupling between global brain signals and ventricles - more frequent in cognitively normal controls - we discovered additional modes where specific brain areas temporarily align with ventricle signals. At the cortical level, these modes form canonical resting-state networks, such as the Default Mode Network, which occurs less in AD or the Frontoparietal Network, which correlates positively with memory scores. The direct link between ventricle and brain signals challenges the common practice of removing CSF components from rsfMRI analyses and questions the origin of cortical signal fluctuations forming functional networks, which may reflect region-specific fluid inflow patterns. These findings provide new insights into the relationship between brain clearance mechanisms and network dysfunction in neurodegenerative diseases.

Zero-phase-delay connectivity increases the reliability, concordance with structure, and prognostic ability of functional connectivity metrics
Zero-phase-delay synchrony between the activity of distant neural populations has been robustly observed. Nevertheless, contemporary electroencephalography and magnetencephalography functional connectivity analyses typically exclude zero-phase-delay functional connections, assuming that they are predominantly artefactual. However, the effects of excluding them on the performance of functional connectivity metrics as potential biomarkers are unknown. Here, we showed that most cortico-cortical functional connections occur with zero- or near-zero phase-delay, even where such connectivity was unlikely to be artefactual. Including, rather than excluding, zero-phase-delay connectivity increased the reliability, concordance with structural connectivity, and predictive validity (for longitudinal changes in cognition) of functional connectivity metrics. We found that excluding zero-phase-delay connections penalised functional connectivity strength between the strongest structurally connected regions: stronger structural connections led to functional connections with phase-delays closer to zero, mediated by a shorter signal propagation time. Our findings challenge generally accepted assumptions that zero-phase-exclusive methods are superior to zero-phase-inclusive methods.

EXPLORING SOCIAL COGNITION DECLINE ACROSS ALZHEIMER'S DISEASE SEVERITY: INSIGHTS FROM A COMPARATIVE ANALYSIS
The detection of subtle behavioral signs in early stages of Alzheimers Disease (AD) is of great importance to start clinical investigation. Memory and reasoning problems are usually more commonly reported by relatives than those associated with Social Cognition (SC). This study focusing on deficits in SC as a behavioral marker of Alzheimers Disease (AD) investigated: (1) selective impairments in SC domains according to stage of disease; (2) if impairments were better explained by nonsocial cognitive dysfunctions. Sixty-five elderly distributed into groups according to the Mini-Mental State Examination (Healthy, Mild cognitive impairment, Mild-AD, Moderate-AD) participated in the study. All underwent the Edinburgh Social Cognition Test (ESCoT), which investigates the cognitive and affective domains of Theory of Mind, as well as interpersonal and intrapersonal understanding of social norms. The nonsocial cognitive battery included traditional tests of episodic memory, executive functions, verbal fluency and language comprehension. Mediation analysis was performed to understand the influence of nonsocial cognitive skills in SC impairment among groups. Results revealed prominent losses in SC in moderate AD regardless of memory-skills. Theory of mind was a crucial link between social and nonsocial cognitive abilities. Executive functioning, verbal fluency and language comprehension mediated group-related cognitive decline's effects on SC impairment. These findings underscore the complex interplay between cognitive domains in AD and suggest that selective impairments of SC highlight is as potential a marker for tracking the progression of the disease.