bioRxiv Subject Collection: Neuroscience's Journal

PyMouse Lifter: Real Time 3-D Pose Estimation for Mice with Only 2-D Annotation Via Data Synthesis
Neural-network based pose estimation models have become increasingly popular for quantitative analysis of mouse behavior, yet most recordings still use a single 2 D camera view and therefore lack the depth cues needed for accurate 3 D kinematics. Existing open source 3 D mouse datasets for training deep-learning models cover only a narrow range of environments and do not generalize well to various laboratory settings. To overcome these limitations, we introduce PyMouseLifter, a pipeline that automatically reconstructs 3 D mouse poses from ordinary 2-D top view videos with minimal manual 2-D annotation. PyMouseLifter combines (i) an anatomically realistic 3 D mouse model for automated data synthesis, (ii) a monocular depth estimation model, and (iii) a 2 D key point estimation model, enabling accurate 3 D reconstruction (model-based 3D inference) in virtually any open field arena without using depth or multiple camera views for reconstruction. We validate the system on multiple datasets against depth camera ground truth and show that the lifted 3D trajectories yield improved behavior classification over 2 D data and can be implemented in real time.

Retrograde Optogenetics Reveals Functional Convergence within a Corticotectal Pathway of Non-Human Primates
Retrograde optogenetics enables tagging and manipulation of pathway-defined neurons, although its application to the non-human primate brain remains challenging. We applied the technique to identify, record, and activate neurons forming the frontal eye field-superior colliculus projection in behaving macaques. Optical stimulation evoked robust contralateral saccades and pathway-selective opto-tagging revealed the functional visuo-motor convergences within this pathway. Our findings resolve a longstanding controversy about the frontal oculomotor outputs and highlight retrograde optogenetics as a powerful tool for primate circuit-level studies.

Habenular μ-opioid receptor knockout and chronic systemic receptor blockade promote negative affect and heighten nociceptive sensitivity
The -opioid receptor (MOR), a subtype of opioid G protein-coupled receptor, is expressed in multiple brain circuits and is particularly enriched in the habenula, a small epithalamic structure implicated in aversive states. MOR dysfunction has been linked to several psychiatric and nociceptive disorders. Identifying the key brain regions mediating the behavioral consequences of disrupted MOR signaling can shed light on the role of the opioid system in mood and pain regulation. In this study, we administered methocinnamox (MCAM), a long-acting, pseudo-irreversible MOR antagonist, acutely or chronically to adult C57BL/6J mice. A comprehensive behavioral battery was used to assess affective, social, and pain behavior. A single MCAM administration (10 mg/kg, s.c.) did not alter baseline behavior, but blocked opioid-induced analgesia, suggesting that basal -opioid tone does not contribute to these behaviors. In contrast, chronic MCAM administration (10 mg/kg, s.c., 3x/week for 4 weeks) led to increased anxiety-like behavior and decreased sociability, as well as enhanced mechanical allodynia and thermal hyperalgesia. Remarkably, selective knockout of habenular MORs in adult Oprm1fl/fl mice reproduced key features of the chronic MCAM phenotype, including anxiety-like behavior and mechanical hyperalgesia. Together, these findings reveal that sustained inhibition of MOR signaling disrupts affective and nociceptive processing and highlight the habenula as a node mediating key behavioral deficits of disrupted opioid signaling.

Ca2+-phospholipid-dependent regulation of Munc13-1 is essential for post-tetanic potentiation at mossy fiber synapses and supports working memory
Hippocampal mossy fiber (hMF) to CA3 pyramidal cell synapses are thought to support the formation of working memory through presynaptic short-term facilitation (STF) and post-tetanic potentiation (PTP). However, the molecular mechanisms underlying these transient forms of synaptic enhancement remain poorly understood. We show here that Munc13-1-mediated priming of synaptic vesicles (SVs) at active zones controls hMF STF and PTP in response to Ca2+-phospholipid and Ca2+-calmodulin (CaM) signaling. Knock-in mice expressing Munc13-1 variants that are insensitive to Ca2+-phospholipid and Ca2+-CaM signaling exhibit severely impaired STF and PTP at hMF synapses. Moreover, the PTP-induction threshold is strongly increased upon the loss of Ca2+-phospholipid-Munc13-1 signaling. Since these synaptic defects are accompanied by working memory deficits, especially in mice expressing the Ca2+-phospholipid-insensitive Munc13-1 variant, we conclude that Ca2+-dependent regulation of Munc13-1-mediated SV priming co-determines hMF short-term plasticity and working memory formation.

Mechanically-gated currents in mouse sensory neurons lacking PIEZO2
Touch sensation starts with the opening of mechanically-gated activated ion channels at neuroglial endings of mechanoreceptors in the skin. The function of around half of low threshold mechanoreceptors is dependent on the presence of the mechanically gated ion channel PIEZO2. It has been reported that particularly rapidly-adapting mechanosensitive currents (RA-currents) in the cell bodies of acutely cultured sensory neurons are dependent on PIEZO2. Here we re-examined this question by making a quantitative study of mechanically-gated currents activated by substrate deflection in sensory neurons lacking PIEZO2. We characterized mechanically-gated currents from embryonic and post-natal sensory neurons, taken from global Piezo2-/- or Piezo2 conditional knockouts (Piezo2cko), respectively. Surprisingly in both models, Piezo2 gene deletion was not associated with any significant reduction in the sensitivity or incidence of mechanosensitive currents compared to wild type controls. There was, however, a moderate reduction in the incidence of RA-currents with very fast activation and inactivation kinetics in both embryonic Piezo2-/- and juvenile Piezo2cKO mice. These results show that PIEZO2 channels are not the only mechanosensitive channels mediating RA-currents in sensory neurons. Furthermore, our data suggest that the phenotypes associated with Piezo2 loss of function alleles may sometimes be due to secondary effects of gene deletion, for example, by changing the developmental trajectory of sensory neurons. Emphasis should be put on the diversity of mechanosensitive ion channel function in sensory neurons which needs to be further elucidated.

Microglia-to-neuron signaling increases lipid droplet metabolism, enhancing neuronal network activity
Microglia regulate neuronal circuit plasticity. Disrupting their homeostatic function has detrimental effects on neuronal circuit health. Neuroinflammation contributes to the onset and progression of neurodegenerative diseases, including Alzheimers disease, with several microglial activation genes linked to increased risk for these conditions. Inflammatory microglia alter neuronal excitability, inducing metabolic strain. Interestingly, expression of APOE4, the strongest genetic risk factor for Alzheimers disease, affects both microglial activation and neuronal excitability, highlighting the interplay between lipid metabolism, inflammation, and neuronal function. It remains unclear how microglial inflammatory state is conveyed to neurons to affect circuit function and whether APOE4 expression alters this intercellular communication. Here, we use a reductionist model of human iPSC-derived microglial and neuronal monocultures to dissect how the APOE genotype in each cell-type independently contributes to microglial regulation of neuronal activity during inflammation. Conditioned media from LPS-stimulated microglia increased neuronal network activity, assessed by calcium imaging, with APOE4 microglial conditioned media driving higher neuronal firing rates than APOE3 conditioned media. Both APOE3 and APOE4 neurons increase network activity in response to conditioned media treatments, while APOE4 neurons uniquely increase presynaptic puncta with APOE4 microglial conditioned media. Conditioned media-derived exosomes from LPS-stimulated microglia can mediate increases to network activity. Lastly, increased network activity is accompanied by increased lipid droplet metabolism and blocking lipid droplet metabolism abolishes network activity. These findings illuminate how microglia-to-neuron communication drives inflammation-induced changes in neuronal circuit function, demonstrate a role for neuronal lipid droplets in network activity, and support a potential mechanism through which APOE4 increases neuronal excitability.

Alpha-Beta oscillations implement inhibition of the ventral attention network during an attention task
Selective attention is a crucial mechanism that allows us to focus on relevant information while ignoring irrelevant information. Although extensive literature has proposed that alpha oscillations (7-14 Hz) suppress distractor processing, recent studies have questioned this role. In this magnetoencephalography (MEG) study, we used a modified Stroop task to investigate whether (1) alpha oscillations are associated with functional inhibition in higher-order visual regions and the ventral attention network (VAN), and (2) alpha phase adjusts in anticipation of relevant and irrelevant stimuli. We found no significant increase in pre-stimulus alpha power or phase adjustment over higher-order visual regions in the attend-color condition, consistent with the absence of a behavioral Stroop effect. However, we observed elevated alpha-beta power (10-20 Hz) in the VAN during both attend-color and attend-word conditions compared to control conditions, occurring before stimulus onset. Higher alpha-beta power in the right temporo-parietal junction correlated with faster reaction times, suggesting that inhibition of this network region facilitates task performance. This alpha-beta modulation in the VAN may represent a general mechanism for resisting distraction, preventing attentional capture by irrelevant information regardless of task condition. Additionally, we found enhanced theta power (4 Hz) over the VAN and left medial frontal gyrus (part of the cognitive control network) in both experimental conditions. Theta power correlated with improved reaction times across these regions. Furthermore, theta activity in the left medial frontal gyrus synchronized with VAN nodes, potentially indicating cross-network interaction between cognitive control and attention systems.

Distinguishing cortical indexes of arousal and awareness in sleeping patients with unresponsive wakefulness syndrome
Consciousness is assumed to be defined by two components: awareness and arousal. However, the cortical indexes for these two components remain unclear. Since patients with unresponsive wakefulness syndrome (UWS) are supposed to have no awareness but arousal, sleep-wake cycle in patients with UWS may reflect pure arousal fluctuations, and then could distinguish the cortical indexes for arousal from ones for awareness. This study recorded nighttime polysomnography in patients with UWS, patients with minimally conscious state (MCS) and healthy controls, showing that spectral slope could index arousal, fluctuating among sleep stages in all three groups. Both spectral entropy and Lempel-Ziv complexity can index awareness, showing a significant difference between conscious and unconscious states. These findings provide fundamental evidence for the two-component hypothesis of consciousness.

Large-scale wearable data reveal spatiotemporal organization of annual sleep patterns
Sleep is fundamental to health, yet large-scale, objective data on how geography shapes sleep behavior remain scarce. We analyzed over 45 million nights of sensor data from 105,741 German adults wearing consumer-grade wearables across 2.7 years. Sleep timing displayed a continuous east-west gradient, with later onset, midsleep, and offset in western regions, consistent with solar progression. This effect was strongest on weekends and in rural areas, where midsleep was delayed by 2.2 minutes per degree longitude and sleep duration increased by 1.0 minute. A north-south gradient also emerged. Weekday midsleep advanced by 0.9 minutes per degree latitude, while weekend midsleep was delayed by 0.2 minutes, resulting in greater social jetlag in the north. Sleep duration declined toward higher latitudes across both day types. Seasonal analyses revealed consistent annual rhythms. Sleep duration increased by 24.7 minutes in winter relative to summer, and sleep offset closely followed sunrise. These patterns highlight the joint influence of solar and social time on sleep, with implications for regionally tailored public health strategies.

Defining and measuring proximity to criticality
Over the half century since the renormalization group (RG) brought about deep understanding of critical phenomena in condensed matter physics, it has been claimed that diverse social, engineered, astrophysical, and biological systems operate close to criticality. However, these systems do not afford the neat phase diagrams and exquisite control available in condensed matter physics. How can one assess proximity to criticality when control parameters are unknown, difficult to manipulate experimentally, and fluctuating in response to changing environmental or internal conditions? Here we meet this challenge with a rigorous theoretical framework and data-analytic strategy for measuring proximity to criticality from observed system dynamics. We developed a temporal RG, well-suited to commonly measured time series, and an information theoretic quantification of proximity to criticality that is independent of model parameterization. After benchmarking our approach on diverse ground-truth cases, we apply it to recordings of spiking activity in the mammalian brain, addressing a long-standing controversy. We show that brain dynamics shift closer to criticality during wakefulness and shift away during deep sleep.

Oxytocin neurons in the anterior and posterior paraventricular nucleus have distinct behavioral functions and electrophysiological profiles
Oxytocin is a neuropeptide that can either promote or inhibit affiliative social behaviors. Recent evidence suggests that these diverse effects are mediated by distinct oxytocin receptor-expressing neuron. An outstanding question is whether these behavioral effects are also driven by distinct or overlapping populations of oxytocin-producing neurons. The paraventricular nucleus (PVN) of the hypothalamus is a major source of oxytocin and sends projections to the mesolimbic dopamine system and the extended amygdala. Previous work found that social defeat increased oxytocin neuron activity in the anterior PVN (aPVN) but not posterior PVN (pPVN). We reduced oxytocin synthesis with antisense morpholino oligonucleotides in either anterior or posterior PVN in California mice (Peromyscus californicus), a strong model system for studying effects of social stress on brain function and behavior. Antisense morpholinos in aPVN had no effect on behavior in unstressed females but increased social approach and reduced social vigilance in females exposed to social defeat stress. In pPVN, antisense morpholinos reduced social approach in unstressed male and female California mice. We then used OxtCre mice to compare electrophysiological profiles of oxytocin in aPVN and pPVN with a population of oxytocin neurons in the bed nucleus of the stria terminalis (BNST). Oxytocin neurons in aPVN and BNST oxytocin neurons had higher post-synaptic potentials and responded more strongly to current injections versus oxytocin neurons in pPVN. These findings shed light onto functional and physiological heterogeneity of PVN oxytocin neurons. Our results suggest that context dependent effects of oxytocin are mediated by different populations of oxytocin neurons.

Unfolding spatiotemporal representations of 3D visual perception in the human brain
Although visual input is initially recorded in two dimensions on our retinas, we perceive and interact with the world in three dimensions. Achieving 3D perception requires the brain to integrate 2D spatial representations with multiple depth cues, such as binocular disparity. However, most studies typically examine 2D and depth information in isolation, leaving the integrated nature of 3D spatial encoding largely underexplored. In this study, we collected a large-scale, multimodal neuroimaging dataset consisting of multiple EEG and fMRI sessions while participants viewed stereoscopic 3D stimuli through red-green anaglyph glasses. Participants first completed a behavioral session including depth judgement tasks and a novel cube adjustment task to quantify and calibrate individual depth perception in units of binocular disparity. Then during two EEG and two fMRI sessions, participants passively viewed stimuli presented at 64 systematically sampled 3D locations, yielding over 66,000 trials in total across ten participants. Combining this large-scale EEG-fMRI dataset with computational methods via representational similarity analysis, we not only systematically characterized the spatiotemporal representation of multiple spatial features in 3D perception but also explored how different coordinate systems (e.g., Cartesian or Polar) might be employed across brain regions and time. Our results reveal that human brains employ multiple types of spatial feature representations and coordinate systems to encode spatial locations at different temporal stages and across distinct cortical regions. In addition to strong representations of 2D space throughout visual cortex, we find unique representations for depth and 3D features in later timepoints and visual areas, including some evidence for 3D processing in parahippocampus. These findings contribute to a more comprehensive understanding of the spatiotemporal organization of neural representations that support 3D perception. Additionally, our novel large dataset will be made openly available to support future research on 3D perception and spatial cognition.

ACTION ENHANCEMENT DRIVES THE EMBODIMENT OF A SUPERNUMERARY ROBOTIC DIGIT
Wearable robotic supernumerary limbs enhance motor capabilities by leveraging principles from both engineering and neuroscience. Despite extensive research, how supernumerary limbs become embodied and the relationship between embodiment and artificially enhanced action possibilities remain largely unexplored. This gap has implications for device design and clinical adoption, as embodiment likely promotes patients' acceptance and daily use of their supernumerary limb. Using an adapted proprioceptive drift paradigm from rubber hand illusion research, we investigated the embodiment of a robotic Soft Sixth Finger (SSF). Participants performed grasp-to-lift actions versus mere lift actions without object interaction, wearing the SSF on two different configurations: the palm and back of the hand. The Palm condition involved achievable actions without the SSF, while the Dorsal condition enabled otherwise anatomically impossible actions, extending beyond the physiological motor repertoire. Hand proprioceptive drift was measured before and after each task. Results showed that the SSF impacted body representation only when dorsally mounted. Proprioceptive drift was significantly higher for grasp-to-lift actions in Dorsal versus Palm conditions and higher for grasp-to-lift versus mere lift in the Dorsal condition only. This proves that SSF embodiment relates to extending, rather than substituting, users' action possibilities. Future research must integrate behavioral, cognitive, and neural measurements across healthy and patient populations. Our study provides an ideal model for clinical application, demonstrating how enabling otherwise impossible actions drives robotic supernumerary finger embodiment.

Musical complexity governs a tradeoff between reliability and dimensionality in the neural code
The rich experience of listening to music depends on the neural integration of its constituent elements within the early auditory pathway. Here, we performed the first large-scale study of neural responses to complex music to characterize the neural coding of individual instruments and mixtures with both normal hearing and mild-to-moderate hearing loss. Using coherence and manifold analyses along with deep learning, we identified strong nonlinear interactions in mixed music that impacted the fidelity and geometry of the neural code. We found that increasing musical complexity resulted in the creation of new neural modes, but this increased dimensionality was associated with decreased reliability. This tradeoff persisted even after hearing loss, the effects of which were largely corrected with suitable amplification. These results suggest that the neural coding of music is governed by an inherent tradeoff and highlight a fundamental challenge in maintaining fidelity while processing sensory inputs with increasing complexity.

Distinct Motor Cortex Interneuron Plasticity and Its Association with Prefrontal Brain Volume in Parkinson's Disease
Parkinson's disease (PD) is characterized by motor and cognitive deficits, including abnormal primary motor cortex (M1) excitability and diminished sensorimotor neuroplasticity. While paired associative stimulation (PAS) can induce M1 plasticity, people with PD (PwPD) demonstrate variability that cannot be accounted for by disease progression or medication status. Distinct M1 interneuron populations and attention-related brain structures may influence the reduced PAS-induced neuroplasticity. We aimed to characterize M1 interneuron plasticity in PwPD using attention-modulated PAS and identify neurostructural correlates. PwPD underwent MRI, then a PAS protocol with task-relevant attention. Transcranial magnetic stimulation (TMS) assessments of corticospinal excitability using posterior-to-anterior (PA) and anterior-to-posterior (AP) current directions were employed before and three post-PAS time-points. PAS induced distinct time-dependent M1 interneuron excitability changes. PA TMS showed increased corticospinal excitability at all post-PAS time-points; AP TMS increased only at 30 minutes. Rostral middle frontal gyrus volume uniquely explained variance in PA-sensitive M1 interneuron plasticity. In contrast, AP-sensitive plasticity was associated with baseline AP TMS excitability and age. These findings highlight that M1 interneuron circuits show unique neuroplasticity patterns in PwPD and relate to prefrontal brain volume. Our results suggest a complex interplay between motor and cognition-related deficits as interrelated pathophysiological features in PD.

Rational Expectations and Kinematic Information in Coordination Games
Successful coordination often requires integrating strategic reasoning with real-time observations of others' actions, yet how humans resolve conflicts between these information sources remains unclear. This study aimed to fill this gap by examining how people coordinate in a strategic game when observing partial kinematic information from their partner's actions. Participants played a HI-LO game with a virtual partner, choosing payoffs based on grasping movements toward invisible large and small targets. Hand movements were presented as schematic animations, with partners grasping targets linked to higher or lower payoffs across two configurations. Participants relied exclusively on kinematic cues from hand shape changes in maximum grip aperture to infer their partner's choices. There were two main findings. While participants preferred higher payoffs consistent with rational game-theoretic expectations, reliable kinematic cues overrode these expectations. When early grip aperture changes indicated the partner was reaching for a large target associated with a lower payoff, participants abandoned their default preference for higher payoffs. They chose the lower option instead, achieving a high coordination success rate. These findings demonstrate that people prioritize kinematic cues about others' choices over theoretical assumptions about rational behavior when coordinating. This suggests that movement-based inferences about others' actions in natural social interactions may be weighted more heavily than strategic reasoning when the two sources of information conflict.