bioRxiv Subject Collection: Neuroscience's Journal

Population-Level Activity Dissociates Preparatory Overt from Covert Attention
The neural signatures of preparing overt eye movements and directing covert spatial attention overlap as they recruit the same brain areas. Yet, these neural signatures are dissociable at the single-cell level: Specific cells within these visuo-oculomotor areas are exclusively involved in either motor preparation or covert attention. Nevertheless, it has been proposed that many cells in visuo-oculomotor areas are involved in both motor preparation and covert attention, and consequently their neural signatures should functionally overlap to a large degree. Here, we put this proposal to the test: we combined EEG with sensitive decoding techniques to investigate whether the neural signatures of preparatory overt and covert attention are dissociable across large-scale neuronal populations. We found that neural decoding reliably discerned whether overt or covert attention was shifted well before saccade initiation. Further, inverted encoding modeling revealed sharper spatially tuned activity in preparatory overt than in covert attention. We then asked whether preparatory overt attention achieved sharper spatially-tuned activity by using "more-of-the-same" covert attention, or by recruiting additional spatially selective neural processing. Cross-decoding results demonstrated that preparatory overt attention recruited at least one additional, frontal process. This additional spatially selective process emerged early and likely reflects motor preparation or predictive remapping. To summarize, we found that the neural signatures of overt and covert attention overlap, yet diverge rapidly, in part because overt attention employs an additional spatially selective neural process. Extending beyond a dissociation on the single-cell level, our findings demonstrate that population-level neural activity dissociates preparatory overt from covert attention.

Empirical evidence of the neuroactive potential of the gut on neurochemistry in the gut-brain axis in humans
The gut microbiome produces a wide range of neuroactive metabolites capable of influencing brain neurotransmitter systems. While preclinical studies suggest these microbial pathways modulate cognitive and emotional processes, evidence in humans remains limited. This study investigates the relationship between gut microbiome-derived neuroactive pathways and in vivo brain neurotransmitter concentrations in healthy young females. Using proton magnetic resonance spectroscopy (1H-MRS), we quantified GABA and glutamate levels in the dorsolateral prefrontal cortex (dlPFC), anterior cingulate cortex (ACC), and inferior occipital gyrus (IOG). Parallel gut microbiome profiling identified functional pathways associated with the synthesis and degradation of GABA, glutamate, short-chain fatty acids, p-cresol, and inositol. We observed significant, region-specific associations between microbial neuroactive pathways and both neurotransmitter concentrations and excitatory/inhibitory (E/I) balance, a key regulator of neuroplasticity and mental health. Notably, microbial GABA synthesis pathways were inversely related to brain glutamate levels in the dlPFC, while glutamate synthesis pathways correlated with GABA levels in the ACC. Additional pathways, including p-cresol and SCFA metabolism, showed region-dependent relationships with brain neurochemistry and E/I ratios. These neurochemical findings were complemented by associations between the same microbial pathways and psychological outcomes, including anxiety, depressive symptoms, and sleep quality. These findings provide new evidence of gut-derived metabolic influence on human brain neurotransmitter dynamics and mental wellbeing, highlighting the gut-brain axis as a promising target for microbiome-based interventions supporting cognitive resilience and emotional health.

Statistical learning drives anticipatory micro-saccades toward suppressed distractor locations
Statistical learning enables individuals to suppress distracting but salient information, yet the mechanisms underlying this suppression remain unclear. While some suggest that suppression is proactive, occurring without first attending to distractor locations, others argue that it is reactive, requiring covert attention toward the distractor location before suppression can occur. To address this fierce debate, the current study recorded micro-saccades--an index of covert attention--during the pre-stimulus interval, alongside electroencephalogram (EEG) data collected during a visual search task. Participants were instructed to ignore a salient distractor that appeared more frequently at one specific location than at others. Learning high-probability distractor locations reduced attentional capture by salient distractors, coinciding with increased micro-saccade rates and decodable representations of these locations in alpha power (8-14 Hz) before stimulus onset. Strikingly, these anticipatory micro-saccades were more frequently directed toward high-probability distractor locations than away from them, supporting a reactive suppression mechanism. Overall, these findings highlight a crucial role for the oculomotor system in encoding and responding to learned spatial regularities.

CaMK2rep: A Highly Sensitive Genetically Encoded Biosensor for Monitoring CaMKII Activity in Mammalian Cells
Accurately monitoring calcium/calmodulin-dependent protein kinase II (CaMKII) activity in cells remains a significant challenge due to the limited sensitivity and narrow dynamic range of existing genetically encoded sensors. Here, we introduce CaMK2rep, a novel phosphorylation-based biosensor that enables robust, specific, and high-sensitivity detection of CaMKII activity. CaMK2rep is designed with two tandem CaMKII consensus sites embedded within the native sequence context of synapsin, and its phosphorylation is detected via a phospho-specific antibody, allowing both biochemical and morphological analyses. We validated CaMK2rep in HeLa cells and cultured hippocampal neurons, demonstrating a near-linear response to CaMKII expression levels and stimulation intensity, and no detectable cytotoxicity. To complement CaMK2rep measurements, we employed the live-cell CaMKAR1 reporter to monitor CaMKII activity dynamics. Using both tools, we investigated the role of neurogranin (Ng), a major postsynaptic calmodulin (CaM) binding protein, and obtained consistent evidence supporting a CaM-buffering model in which Ng limits basal CaMKII activation by sequestering CaM. These findings establish CaMK2rep as a sensitive, specific, and versatile biosensor for CaMKII signaling, particularly well-suited for immunoblot-based population analyses. They also illustrate the value of combining orthogonal genetically encoded tools to interrogate complex signaling mechanisms in both physiological and pathological contexts.

An intercellular signaling pathway in the mouse retina connects Kv2.1, GLT-1, and nitric oxide synthase 1 to optic nerve regeneration
We report here that a multicellular signaling pathway in the mouse retina targeting glutamate homeostasis and nitric oxide production is activated upon optic nerve injury and modulates retinal ganglion cells' (RGCs') ability to mount a robust regenerative response. A novel highly sensitive and specific NO sensor (FL2) revealed that optic nerve injury leads to a rapid, prolonged NO elevation in the inner retina. Amacrine cell-specific genetic deletion of the neuron-specific isoform of nitric oxide synthase (NOS1, nNOS) or the NOS1 inhibitor N(w)-propyl-L-arginine (L-NPA) suppressed optic nerve regeneration. Steps leading to NOS1 activation are shown to include Kv2.1 phosphorylation and activation, reversal of glutamate uptake by the glutamate transporter GLT-1 (EAAT2), and subsequent NMDA receptor activation. Conditional knockout of the glutamate transporter GLT-1 in bipolar cells, intraocular injection of the GLT-1 blockers dihydrokainate (DHK) or WAY213613, the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5), or the Kv2.1 blockers RY796 or stromatoxin all suppressed NO generation and strongly diminished RGCs' ability to respond to Pten deletion and other factors. Thus, optic nerve injury activates a sequence of pathophysiological changes in retinal interneurons that gates RGCs' ability to regenerate injured axons.

Cholinergic blockade reveals role for human hippocampal theta in encoding but not retrieval
Cholinergic dysfunction is a hallmark of Alzheimer's disease and other memory disorders. Yet, the neurophysiological mechanisms linking cholinergic signaling to memory remain poorly understood. In this study, we administered scopolamine, a muscarinic cholinergic antagonist, to neurosurgical patients with intracranial electrodes as they performed an associative recognition memory task. When scopolamine was present at encoding, we observed disruptions to hippocampal slow theta oscillations (2-4 Hz), with selective impairments to recollection-based memory. However, when scopolamine was present during retrieval alone, we observed disruptions to slow theta without impaired memory performance. These disruptions included dose-dependent reductions in theta power, theta phase reset, and encoding-retrieval pattern reinstatement. Together, our results challenge the notion that theta oscillations are necessary for memory retrieval, and instead suggest that theta universally reflects an encoding-related neural state. These findings motivate updates to current models of acetylcholine's role in memory and may inform future therapies targeting rhythmic biomarkers of memory dysfunction.

Developmental embedding of parvalbumin interneurons drives local and crosshemispheric prefrontal gamma synchrony
Gamma oscillations are a pivotal trait of cortical cognitive processing. However, the ability to generate gamma oscillations evolves with age and requires cellular adjustments of the underlying neural networks. In the prefrontal cortex, gamma oscillations emerge relatively late compared to other cortical areas, yet the developmental mechanisms leading to the generation of adult-like gamma oscillations are poorly understood. Here, we combine bilateral in vivo electrophysiology and selective optogenetic manipulations of parvalbumin- (PV+) and somatostatin-positive (SOM+) interneurons in the mouse medial prefrontal cortex along late development to investigate their role for the age-dependent maturation of gamma oscillations. We show that crosshemispheric gamma synchrony strengthens with age, in line with the previously reported increase in local gamma power. Following a similar timeline, the inhibitory effect of PV+ interneurons emerges which start to functionally operate within the classical gamma frequency range from adolescence onwards. In contrast, SOM+ interneurons show no such age-dependent functional integration and display their beta oscillation modulating inhibitory effect across age. These data identify the SOM+ to PV+ interneuron switch as a mechanism of gamma ontogeny and emergence of crosshemispheric synchrony in the developing prefrontal cortex.

22q11 deletion selectively alters progenitor states and projection neuron identities in the developing cerebral cortex
Heterozygous deletion of multiple contiguous genes associated with 22q11.2 Deletion Syndrome (22q11DS), a developmental disorder with significant risk for autistic spectrum disorder (ASD) and schizophrenia (Scz), selectively compromises neurogenic capacities of a temporally distinct cohort of cerebral cortical basal progenitors (bPs), prefiguring diminished frequency, divergent times of origin, positions, and identities of a subset of Layer 2/3 projection neuron (PN) progeny in the LgDel 22q11DS mouse model. LgDel bPs express 24/28 contiguous murine 22q11 gene orthologues at diminished levels; in parallel, cell cycle kinetics, modes of division, gene expression levels, and DNA methylation states are aberrant in LgDel bPs but not their apical progenitor precursors. Accordingly, targeted disruption of bP proliferative and transcriptional states selectively alters Layer 2/3 PN identities and frequencies, prefiguring atypical association cortico-cortical connections and behavioral deficits associated with ASD and Scz pathology in a mouse model of 22q11DS.

Cerebellar tDCS differentially modulates sensory inputs in somatosensory cortex and cerebellum.
Cerebellar transcranial direct-current stimulation (Cb-tDCS) is a promising tool for non-invasive modulation of cerebellar activity and has been proposed for the treatment of cerebellum-related disorders. However, how external currents applied to the cerebellum affect local and distant circuits remains unclear. In this study, we examined the immediate and aftereffects of Cb-tDCS on sensory inputs recorded in the cerebellar Crus I/II and primary somatosensory cortex (S1) in response to whisker stimulation. We also assessed changes in the excitation/inhibition balance using vGLUT1 and GAD 65-67 immunoreactivity. Anodal and cathodal Cb-tDCS respectively induced an immediate increase and decrease in the trigeminal component in Crus I/II but no aftereffects were observed 20 minutes post-stimulation. In S1, Cb-tDCS resulted in polarity-dependent modulation of the N1 component during stimulation, which was opposite to the changes induced in Crus I/II and a sustained increase after anodal Cb-tDCS, accompanied by reduced GAD 65-67 immunoreactivity. While power spectrum analysis revealed no changes in Crus I/II, cathodal Cb-tDCS significantly modulated gamma (30-45 Hz) and high-frequency oscillations (255-300 Hz) in S1. These findings indicate that Cb-tDCS can modulate sensory inputs during stimulation and exert delayed effects in distant cortical areas, emphasizing the need to consider both online and remote network modulation in clinical applications.

Non-invasive mapping of the temporal processing hierarchy in the human visual cortex
Vision is not instantaneous but evolves over time. However, simultaneously capturing the fine spatial details and the rapid temporal dynamics of visual processing remains a major challenge, resulting in a gap in our understanding of spatiotemporal dynamics. Here, we introduce a forward modeling technique that bridges high-spatial resolution fMRI with high-temporal resolution MEG, enabling us to non-invasively measure different levels of the visual hierarchy in humans and their involvement in visual processing with millisecond precision. Using fMRI, levels of the visual hierarchy were identified by measuring individuals population receptive fields and determining visual field maps. We predicted how much the activity patterns in each visual field map would contribute to brain responses measured with MEG. By comparing these predicted responses with the measured MEG responses, we assessed how much a given visual field map contributed to the measured MEG response, and, most importantly, when. We combined information from all MEG sensors and revealed a cortical processing hierarchy across visual field maps. We validated the method using cross-validations and demonstrated that the model generalized across MEG sensor types, stimulus shapes, and was robust to the number of visual field maps included in the model. We found that the primary visual cortex captured most of the variance in the MEG sensors and did so earlier in time than extrastriate regions. We also report a processing hierarchy across extra-striate visual field maps and clusters. We effectively combined the advantages of two very different neuroimaging techniques, opening avenues for answering research questions that require recordings with high spatiotemporal detail. By bridging traditionally separate areas of research, our approach helps close longstanding gaps in our understanding of brain function.

Mapping Functional Homologies Between Human and Marmoset Brain Networks Using Movie-Driven Ultra-High Field fMRI
Naturalistic stimuli, such as movies, offer a powerful tool for probing functional brain organization across species. Using movie-driven functional magnetic resonance imaging (md-fMRI), we recorded brain activity in humans and awake marmosets exposed to the same dynamic audiovisual stimulus. We applied tensor independent component analysis (tICA) to identify functional networks in each species, hierarchically clustered these components, and examined their within- and between-species temporal correlations to assess functional homologies. We found strong interspecies correspondence in core sensory networks, particularly those involved in visual and auditory processing, suggesting conserved mechanisms for sensory integration. In contrast, networks associated with higher-order cognition, including prefrontal and temporoparietal areas, were observed primarily in humans, highlighting species-specific specializations. These findings demonstrate the value of naturalistic paradigms and data-driven approaches in revealing both shared and divergent brain network architectures. By openly sharing our data and analysis pipelines, we aim to support future comparative studies and advance the marmoset as a model for investigating the evolutionary foundations of brain function.

Alcohol impacts an fMRI marker of neural inhibition in humans and rodents.
Acute alcohol consumption leads to cognitive and behavioral disinhibition that increase health and social risks, such as traffic fatalities and violence. Although rodent studies have shown that alcohol affects neural inhibition, its relevance to humans remains largely unexplored. Here, we used the Hurst exponent, an fMRI-based marker of neural inhibition, to examine alcohol's effects in both rats and humans. In rats, acute alcohol administration significantly reduced the cortical Hurst exponent, suggesting decrease of neural inhibition. This reduction was strongly correlated with the spatial distribution of GABAA receptor expression, highlighting the key role of these receptors in mediating alcohol's effects. Similarly, in humans, acute alcohol consumption reduced the cortical Hurst exponent, especially in brain regions with high GABAA receptor expression. Our findings provide cross-species in vivo evidence that acute alcohol consumption modulates neural inhibition, offering new insights into the neural mechanisms underlying alcohol-induced behavioral modulation.

BrainVAE: Exploring the role of white matter BOLD in preclinical Alzheimer disease classification
INTRODUCTION: Like gray matter (GM), white matter (WM) BOLD functional signals change in preclinical AD. However, the potential of WM BOLD for identifying preclinical AD remains underexplored. METHODS: We developed BrainVAE, a transformer based variational autoencoder with interpretability, to classify preclinical AD and normal controls using resting state fMRI data. We benchmarked BrainVAE against nine alternative models under three input configurations: WM only, GM only, and combined WM+GM. Interpretability analysis was also performed to investigate each brain regions contribution to the classification task. RESULTS: BrainVAE outperformed other models and performed well (accuracy = 83.42%, F1-score = 91.62%, AUC = 64.50%) using the combined input compared to WM-only and GM-only. Specific WM bundles, including corpus callosum, fornix, and corticospinal tract, were among the most influential features contributing to the classification. DISCUSSION: Incorporating WM BOLD signals improves the distinction of preclinical AD from controls, underscoring the potential role of WM BOLD features for detecting early-stage AD.

Longitudinal monitoring of developmental plasticity in the mouse auditory cortex
The postnatal brain undergoes substantial plasticity in order to represent features and statistics of the sensory world. To date, for technical reasons it has been difficult to examine experience-dependent changes over the course of postnatal development. Here we perform longitudinal two-photon calcium imaging of hundreds of neurons in the auditory cortex of young mice, from postnatal day (P) 12 into adulthood. Auditory cortical neurons started responding to tonal stimuli by P13-14 with an initial tonotopic organization that expanded over the next few days. We documented the daily variation in tuning curves and best frequency, and while some neurons gradually changed their frequency representation across days, altogether our findings support the functional maturation of a largely stable auditory map at the population level. We then compared the representation of mouse pup ultrasonic vocalizations to pure tone responses. Ultrasonic pure tone tuning developed with a delay, coinciding with emerging responses to ultrasonic vocalizations. Vocalization responses initially were observed in neurons tuned to ultrasonic frequencies, but over later development the vocalization responses became independent from ultrasonic frequency tuning. Our results show how varied sensory representations at the single-cell and population levels in the postnatal auditory cortex emerge and change over the course of early-life development.

Individual differences in fear memory expression engage distinct functional brain networks
Fearful stimuli elicit a mix of active (e.g., evasion) and passive (e.g., freezing) behaviors in a wide range of species, including zebrafish (Danio rerio). However, it is not clear if individual differences in fear responses exist and, if so, what parts of the brain may underlie such differences. To probe these questions, we developed a contextual fear conditioning paradigm for zebrafish that uses conspecific alarm substance (CAS) as an unconditioned stimulus where fish associate CAS administration with a specific tank. To identify individual differences, we collected behavioral responses from over 300 fish from four different strains (AB, TU, TL, and WIK) and both sexes. We found that fear memory behavior fell into four distinct groups: non-reactive, evaders, evading freezers, and freezers. We also found that background strain and sex influenced how fish respond to CAS, with males more likely to increase evasive behaviors than females and the TU strain more likely to be non-reactive. Finally, we performed whole-brain activity mapping to identify the brain regions that are associated with different behavioral responses. All groups exposed to the tank had strong engagement of the telencephalon, whereas regions beyond the telencephalon distinguished behavioral groups: animals that have high levels of freezing, but low levels of evasion, uniquely engage the cerebellum, preglomerular nuclei, and pretectal areas, whereas those fish that mix evasion with freezing engage the preoptic and hypothalamic areas. Taken together, these findings reveal that zebrafish exhibit individual differences in fear memory expression that are supported at the neural level by extra-telencephalic regions.

Temporal coding carries more stable cortical visual representations than firing rate over time
The brain's ability to stably represent recurring visual scenes is crucial for behavior. Previous studies have used slow dynamic (1-5 seconds) rate code measurements to study visual tuning, revealing varying degrees of gradual activity changes over time or "representational drifts." However, it remains unclear if there is an underlying neural code that maintains the encoding of information stable over time. In this study, we extracted structures in fast (tens of milliseconds) temporal responses and explored the role of such temporal codes in supporting the stability of visual representations. We tracked the spiking activity of the same visual cortical populations in male mice for 15 consecutive days using custom-developed, large-scale, ultraflexible electrode arrays. Across various types of stimuli, we found that neurons exhibited varying degrees of day-to-day stability in their firing rate-based tuning. The across day stability correlated with tuning reliability. Notably, accounting for spiking temporal dynamics increased single neuron tuning stability, especially for less reliable neurons. Temporal coding further improved population representation discriminability and decoding accuracy. The stability of temporal codes was more correlated with network functional connectivity than rate coding. These results show that temporal coding is crucial for stably encoding sensory stimuli, suggesting its significant role in ensuring consistent sensory experiences.

Changes in slow oscillations and sleep spindles by auditory stimulation positively correlate with memory consolidation in children with epilepsy and controls
Background: Sleep-dependent memory consolidation is supported by sleep spindles during stages 2 and 3 non-rapid eye movement sleep. Sleep spindles and sleep-dependent memory consolidation are both decreased in Rolandic epilepsy (RE). Non-invasive auditory stimulation evokes SOs and SO-spindle complexes in healthy adults but the impact on memory consolidation has been inconsistent. Objective: We investigated the effects of auditory stimulation during sleep on SOs, SO-spindle complexes, and sleep-dependent memory consolidation in children with RE and controls. Methods: A prospective cross-over study was conducted in children with RE and control. Children completed two nap visits with auditory or sham stimulation. SOs and SO-spindle complexes rates were measured offline using validated detectors. Sleep-dependent memory consolidation was assessed using the motor sequence typing task. Results: Auditory stimulation evoked SOs and SO-spindle complexes broadly with maximal effect over frontal electrodes. Compared to sham, stimulation delivered during background activity evoked SOs (29.8% increase, p<0.001) and SO-spindle complexes (16.8% increase, p<0.001) and stimulations delivered near the peak of an ongoing SO upstate maximally evoked SOs (51.3% increase, p<0.001) and SO-spindle complexes (32.3% increase, p<0.001). Changes in frontal SO (1.9% improvement per increase in SO/min; p<0.001) and SO-spindle complexes (9.5% improvement per increase in SO-spindle/min) event rates due to auditory stimulation positively predicted changes in sleep-dependent memory consolidation. Conclusion: Auditory stimulation reliably modulates sleep oscillations when delivered on background activity and during the upstate of SOs. As increased event rates improve memory consolidation, stimulation paradigms to increase SO and SO-spindle complex rates are required to enhance memory.

A protective role of REM sleep in the pathological basal ganglia-cortical circuit of Parkinson's disease
Sleep disturbance is involved in the progression of neurodegenerative diseases, but the neurophysiological mechanisms remain unclear in humans. We recorded sleep at home using portable polysomnography for multiple nights in patients with Parkinson's disease implanted with sensing DBS, capturing intracranial neural signals from both the basal ganglia and motor cortex. We found that longer duration and shorter latency of rapid eye movement (REM) sleep predicted reduced next-morning neural signatures known to be pathological, including resting beta oscillations and hyper-neural connectivity between basal ganglia and motor cortex. Moreover, within REM sleep, stronger cortical delta activity particularly predicted reduced pathological neural signatures. Computational modeling of the evoked potential response in the basal-cortical circuit revealed that REM delta activity was associated with not only the amplitude of the evoked response, but also its natural oscillatory and decaying properties, suggesting REM sleep-mediated neural plasticity. The anti-relationship between delta activity and morning pathological neural signals was found to be primarily driven by the amplitude of delta waves, and less by their occurrence rate. In addition, as the REM sleep percentage and REM delta power increased in later hours during sleep, their protective effects also increased. Overall, our findings highlight the potential protective roles of REM sleep and its associated slow-wave activity in the neurophysiological health of Parkinson's disease.

Neuropeptide signalling and perineurial barrier generate a persistent stress-induced internal state in Drosophila
Although fear conditioning has elucidated cue-evoked acute fear responses, the mechanisms by which stress experiences induce generalized internal states linked to anxiety are poorly understood. Here, we report that robust stress induces a persistent behavioral change characterized by avoidance of a confined space, claustrophobia-like behavior in Drosophila. Unlike aversive memory formation, the development of claustrophobia-like behavior does not require dopamine receptors. Our neuronal screening determined that neuropeptide signalling via Allatostatin-A inactivates the downstream neurons via its receptor AstA-R1, causally inducing claustrophobia-like behavior. Moreover, gene expression profiling of individual fly heads revealed that immune response activation in perineurial barrier is involved in claustrophobia-like behavior. Our data demonstrate that stress-induced persistent behavioral change would not be related to a canonical mechanism of aversive memory formation, rather involves neuropeptidergic signalling and perineurial barrier, providing the mechanism determining internal states which persistently change behavioral modes.

Thermal cycling stimulation via nasal inhalation attenuates Aβ25-35-induced cognitive deficits in C57BL/6 mice
Alzheimer's disease (AD) continues to pose a significant public health challenge, with current treatments demonstrating limited effectiveness, partly due to the difficulty of delivering therapeutics across the blood-brain barrier (BBB). The nose-to-brain (N-2-B) pathway offers a promising alternative, enabling pharmacological agents to circumvent the BBB. However, to date, no drugs have been successfully implemented in clinical settings for the treatment of AD via this route, underscoring the necessity for additional research in this area. Mild stress is thought to activate intrinsic protective mechanisms against neurodegeneration, but traditional methods of inducing stress often lack both specificity and practicality. To address this, we propose inhaling mildly heated air as thermal stimulation, leveraging the N-2-B pathway to elicit mild stress and stimulate the brain. Furthermore, this study employs our thermal cycling hyperthermia (TC-HT) concept, adapted as thermal cycling stimulation via nasal inhalation (TCSNI), which delivers cyclic stimulation to sustain pathway activity while minimizing thermal damage. In this study, we evaluated the health and olfactory effects of TCSNI in C57BL/6 mice, observing no adverse impact. In experimental groups administered with {beta}-amyloid (A{beta}), TCSNI treatment resulted in significant enhancements in cognitive performance as evidenced by Y-Maze and novel object recognition (NOR) assessments, suggesting an improvement in cognitive function. Protein analyses conducted on the hippocampus of the mice indicated a reduction in A{beta} accumulation, alongside increased expression of heat-shock protein 70 (HSP70) and insulin-degrading enzyme (IDE) expression, as well as elevated levels of phosphorylated Akt (p-Akt). These results suggest that N-2-B-delivered TCSNI effectively modulates protein expression and enhances cognitive function, highlighting its potential for further exploration in AD treatment.

The contribution of the locus coeruleus-norepinephrine system to the coupling between pupil-linked arousal and cortical state
Understanding how pupil-linked arousal couples with cortical state is crucial for uncovering the neural mechanisms underlying brain state-dependent cognitive and sensory processing. Pupil size fluctuations reflect rapid changes of the pupil-linked arousal system, indexing brain states as well as the activity of neuromodulatory systems, including the locus coeruleus-norepinephrine (LC-NE) system. We investigated the relationship among phasic pupil dilation, cortical state, and neuromodulation by combining optogenetic LC stimulation with EEG recordings and pupillometry in awake mice. A comparison between EEG signals during spontaneous phasic pupil dilation and those during phasic pupil dilation evoked by LC stimulation revealed distinct EEG power spectrums, with LC activation driving strong modulation in the alpha and beta bands. Using machine learning techniques, we trained a convolutional neural network classifier to distinguish between types of pupil dilation based on the power dynamics of individual EEG frequency bands. The results confirmed that EEG features in the alpha, beta, and gamma bands differ markedly between spontaneous phasic arousal and LC stimulation-evoked arousal. Moreover, pharmacological manipulations to either block or {beta} adrenergic receptors or agonize -2 adrenergic receptors were employed to explore how adrenergic receptors could influence the coupling between phasic pupil dilation and cortical state. With each manipulation uniquely modulating EEG power and pupil size, our results highlight the differentiated role of adrenergic receptors in the maintenance of coupling between pupil-linked arousal and cortical state. This study provides new insights into the complex relationship between pupil-linked arousal and cortical arousal state, underscoring the significant role of the LC-NE system in influencing these arousal states.

The role of effort in adaptation to split-belt locomotion
In many motor learning tasks, the process of error reduction is mirrored in a process of effort reduction, where metabolic cost and or muscle co-contraction decrease gradually in tandem with error. Effort reduction may be incidental to the learning process, but high error movements can be more effortful and a drive to minimize effort costs could also play a role in motor learning. In this study, we focused on the effort requirements of the task, asking whether task (background) effort cost affects learning, aftereffects, or relearning in a split-belt walking task. We hypothesized that greater effort costs would amplify the need to reduce effort and accelerate learning. Alternatively, we hypothesized that greater effort requirement could compromise and slow the learning process. Participants in high, low, and control effort groups completed a split-belt walking task while wearing a weighted vest with 15% body mass, 5% body mass, or the vest only, and we assessed step length asymmetry throughout the protocol. Step length asymmetry changed similarly between groups, with similar rates and extent of learning and relearning and similar patterns of aftereffects when the split-belt perturbation was removed. We found no significant effect of task effort on the process of split-belt adaptation, suggesting that the brain s response to gait asymmetry and ability to adapt to novel dynamics is relatively unchanged by background effort requirements of the task.

A Novel Framework for Quantitative Analysis of Neuronal Primary Cilia in Brain Tissue
Background: Accurate analysis of neuronal primary cilia is essential for understanding developmental processing of neurons. But existing image segmentation methods struggle with staining variability and background noise. To address this, we developed a more robust segmentation and statistical analysis pipeline using an animal model small sample size and with known neuronal microstructure alterations. Methods: Maternal obesity was induced in mice via a high-fat/high-sucrose diet. Hippocampal tissue from 6-month-old offspring of obese and control dams was analyzed. We developed a MATLAB-based pipeline to segment neuronal cilia from z-stack images, applying mathematical transformations and using the Weibull distribution and Bayesian Information Criterion (BIC) to assess group differences Results: The technique segmented cilia despite artifacts, revealing group-specific patterns. Statistical analysis confirmed significant differences, highlighting the method's robustness over traditional tests, especially with small samples. Conclusion: Our method reliably segments neuronal primary cilia in immune-stained sections with thionin-counter staining and offers a sensitive, assumption-free alternative to traditional statistical tests, ideal for small-sample neurobiological studies.