bioRxiv Subject Collection: Neuroscience's Journal

Putting causality into context: Causal capture escapes the visual adaptation of causality
Results from psychophysical studies using visual adaptation suggest that launch detectors in the visual system underlie the perception of causality in simple visual events. These detectors respond to events in which one stimulus collides with another stimulus (i.e., a launch), and do not respond to events where one stimulus passes over another (i.e., a pass). Prolonged visual adaptation to launches significantly reduces observers' propensity to see causal launches at the same retinotopic location. This finding could be taken to indicate that launch detectors are necessary for the local detection of causal launches. However, contextual events that are spatially separated from the test event location shift observers' perception of a causal relation in the direction of the type of contextual event (Scholl & Nakayama, 2002), providing evidence for spatial integration beyond a specific retinotopic location. Here, we used visual adaptation as a tool to investigate whether the contextual influence on causal perception relies on local launch detectors. Before and after adaptation, we determined the proportion of reported launches in ambiguous test events in the presence of no context, launch context, and pass context events. We hypothesized that if the contextual influence relies on (unadapted) local launch detectors, then visual adaptation should affect the contextual influence on causal perception. Before adaptation, a launch-context event increased the proportion of reported launches (while a pass context event decreased it). Visual adaptation to launches significantly decreased the proportion of reported launches in no-context trials, but did not affect perceptual reports in no-context trials. In fact, contextual influences, expressed relative to no-context trials, emerged strongly after adaptation. This result suggests that context effects override strong negative aftereffects from adaptation, indicating that contextual influences operate at a level that bypasses the local launch detector at the adapted location.

Optical activation of midbrain dopamine neurons: do high and low stimulation frequencies produce functionally different effects?
Millard et al. propose that 20- and 50-Hz stimulation of optically excitable, midbrain dopamine neurons triggers functionally distinct effects: The "physiological" firing elicited by a 20-Hz train mimics a reward-prediction error (RPE) whereas the "supra-physiological" firing induced by a 50-Hz train creates "a sensory event that acts as a reward in its own right." Only the 50-Hz trains supported vigorous responding in the face of elevated response costs and sufficed to produce reward-specific Pavlovian-Instrumental Transfer (PIT). Nonetheless, the 20-Hz trains did produce unblocking. Here, we propose a more parsimonious account of these findings that is consistent with the results of prior experiments on operant responding for rewarding optical or electrical stimulation and with a new theory of the role of dopamine signaling in associative conditioning: 1) The effects of the 20- and 50-Hz trains differ quantitatively rather than qualitatively. They are mapped onto a single dimension, reward intensity, which reflects the aggregate release of dopamine in the critical terminal field(s). 2) The non-linear dependence of operant responding on both the aggregate dopamine release and the response cost can explain why responding for the 20- and 50 Hz trains diverged as response cost grew and predicts that this divergence can be eliminated by increasing the number of dopamine neurons stimulated. 3) Terminal-field dopamine concentration provides no information about pulse frequency per se. 4) Operant responding, PIT, and unblocking all depend on reward intensity, but the form and parameters of this dependence can differ, thus generating the observed contrasts in the impact of the 20- and 50-Hz trains. 5) The tested range of experimental conditions is too narrow to specify what constitutes "physiological" firing.

Cryopreservation of Human Cortical Organoids Using Vitrification.
Cryopreservation at ultra-low temperatures is a valuable tool for preserving cells and tissues used in research. However, few protocols exist for the preservation of brain organoid models. Current methods for preserving human cortical organoids (hCOs) rely on conventional slow cooling approaches with organoids suspended in a medium containing a cocktail of cryoprotectants. In contrast, we have optimized a vitrification technique previously used to cryopreserve human embryos and oocytes for application to hCOs. We have successfully cryopreserved hCOs that were generated by two different protocols. The vitrified organoids demonstrate a growth rate, cytoarchitecture, cell type composition and electrical activity comparable to non-vitrified controls. Our hCO cryopreservation method provides a useful alternative approach for bio-banking and cross-institutional collaboration using cortical organoids as their model system.

Conjugated GLP-1 and Estradiol Treatment as a Novel Treatment for Age-related Cognitive Decline in Males and Females
Middle age represents a critical window for metabolic and cognitive health, particularly in the context of rising obesity and diabetes rates. Glucagon-like peptide-1 (GLP-1)-based therapies, which regulate blood glucose and body weight, show sex-specific effects, with estradiol potentiating their metabolic benefits. However, research on GLP-1s cognitive and neuroprotective roles has largely been conducted in males. Here, we investigated the effects of GLP-1 conjugated to estradiol (GE2) on metabolism, cognition, inflammation and neurogenesis in the hippocampus of middle-aged male and female rats fed a standard (SD) or Western (WD) diet. In both sexes, WD increased body weight, and plasma leptin levels. Furthermore, GE2 treatment led to weight loss, enhanced cued and contextual fear memory, reduced inflammation in the hippocampus in SD rats and increased neurogenesis in the dorsal hippocampus. Sex-specific differences were observed in fat distribution, glucose regulation, central inflammation and neuroplasticity after WD and GE2 treatment. In females only, GE2 reduced visceral (gonadal) fat, reduced inflammation in the dorsal hippocampus and improved basal blood glucose in response to a WD. In males only, GE2 normalised reduced hippocampal neurogenesis after a WD and reduced inflammation in the amygdala. These findings suggest that although WD increased body weight and GE2 improved associative learning in both sexes, both WD and GE2 had differential affects on metabolic hormones, insulin regulation, inflammation and neuroplasticity. Our findings underscore the importance of sex-specific approaches in metabolic and neuroprotective therapeutics in middle-age.

Prevention of Transgene Silencing During Human PluripotentStem Cell Differentiation
While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occurs with both randomly integrated transgenes, introduced via transposase or lentiviral methods, and with transgenes targeted to specific genomic sites, including at commonly used safe harbor loci. The challenge to robustly express experimental transgenes in differentiated pluripotent stem cells is a major bottleneck in the field for applications such as CRISPR screening. Here, we conducted a comparative analysis to systematically evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of promoters and transcriptional stability elements are able to prevent transgene silencing during differentiation, whereas chromatin insulators had less impact on silencing and three novel safe harbor integration sites performed similarly to the CLYBL locus. Guided by these insights we developed the PiggyBac vector TK4, which showed complete resistance to transgene silencing across various neuronal and microglial differentiation protocols from six different pluripotent stem cell lines, as independently confirmed by seven different laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation, and holds the potential for broad applications in various research fields.

Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation
In mice, postnatal endothelial cell (EC)-specific knockout of the genes coding for Transforming Growth Factor-Beta Receptor (TGFBR)1 and/or TGFBR2 eliminates TGF-beta signaling in vascular ECs and leads to distinctive central nervous system (CNS) vascular phenotypes. Knockout mice exhibit (1) reduced intra-retinal vascularization, (2) choroidal neovascularization with occasional anastomoses connecting choroidal and intraretinal vasculatures, (3) infiltration of diverse immune cells into the retina, including macrophages, T-cells, B-cells, NK cells, and dendritic cells, (4) a close physical association between immune cells and retinal vasculature, (5) a pro-inflammatory transcriptional state in CNS ECs, with increased ICAM1 immunoreactivity, and (6) increased Smooth Muscle Actin immunostaining in CNS pericytes. Comparisons of the retinal phenotype with two other genetic models of retinal hypovascularization - loss of Norrin/Fzd4 signaling and loss of VEGF signaling - shows that the immune cell infiltrate is greatest with loss of TGF-beta signaling, more modest with loss of Norrin/Fzd4 signaling, and undetectable with loss of VEGF signaling. The phenotypes caused by loss of TGF-beta signaling in ECs recapitulate some of the cardinal features of retinal and neurologic diseases associated with vascular inflammation. These observations suggest that therapies that promote TGF-beta-dependent anti-inflammatory responses in ECs could represent a promising strategy for disease modulation.

Rapid prey capture learning drives a slow resetting of network activity in rodent binocular visual cortex
Neocortical neurons possess stable firing rate set points to which they faithfully return when perturbed. These set points are established early and are stable through adulthood, suggesting they are immutable. Here we challenge this idea using an ethological vision-dependent prey capture learning paradigm in juvenile rats. This learning required visual cortex (V1), and enhanced tuning of V1 neurons to specific behavioral epochs. Chronic recordings revealed a slow, state-dependent increase in V1 firing that began after learning was complete and persisted for days. This upward firing rate plasticity was gradual, gated by wake states, and in L2/3 was driven by a TNF-dependent increase in excitatory synapses onto pyramidal neurons, all features of homeostatic plasticity within V1. Finally, TNF inhibition after learning reduced retention of hunting skills. Thus, naturalistic learning in juvenile animals co-opts homeostatic forms of plasticity to reset firing rate setpoints within V1, in a process that facilitates skill consolidation.

The molecular and cellular underpinnings of human brain lateralization
Hemispheric specialization is a fundamental characteristic of human brain organization, where most individuals exhibit left-hemisphere dominance for language and right-hemisphere dominance for visuospatial attention. While some lateralized functions are evident in other species, the human brain displays a strong, species-wide bias. Despite the evolutionary and functional significance of these asymmetries, their molecular and cellular foundations remain poorly understood. Here, we identify key neurochemical and cellular asymmetries that underpin cortical lateralization. Specifically, we demonstrate lateralized gradients in neurotransmitter receptor densities, particularly along the acetylcholine-norepinephrine axis, as well as asymmetries in mitochondrial distribution and the spatial prevalence of microglia and glutamatergic excitatory neurons. Using a multimodal approach that integrates in vivo functional MRI, PET imaging, and post-mortem transcriptomic and cellular data, we delineate two distinct cortical clusters: a left-lateralized network centered on language processing and a right-lateralized network supporting visuospatial attention. These results highlight a biologically embedded substrate for lateralized cognition that may inform both evolutionary theory and our mechanistic understanding of neuropsychiatric illnesses characterized by disrupted lateralization.

A common framework for semantic memory and semantic composition
How the brain constructs meaning from individual words and phrases is a fundamental question for research in semantic cognition, language and their disorders. These two aspects of meaning are traditionally studied separately, resulting in two large, multi-method literatures, which we sought to bring together in this study. Not only would this address basic cognitive questions of how semantic cognition operates but also because, despite their distinct focuses, both literatures ascribe a critical role to the anterior temporal lobe (ATL) in each aspect of semantics. Given these considerations, we explored the notion that these systems rely on common underlying computational principles when activating conceptual semantic representations via single words, vs. building a coherent semantic representation across sequences of words. The present study used magnetoencephalography and electroencephalography to track brain activity in participants reading nouns and adjective-noun phrases, whilst integrating conceptual variables from both literatures: the concreteness of nouns (e.g., "lettuce" vs. "fiction") and the denotational semantics of adjectives (subsective vs. privative; e.g., "bad" vs. "fake"). Region-of-interest analyses show that the left, and to a lesser extent the right, ATLs responded more strongly to phrases at different timepoints, irrespective of concreteness, suggesting a unified ATL function for semantic memory and composition. Decoding analyses further revealed a time-varying representational format for adjective semantics, whereas representations of noun concreteness were more stable and maintained for around 300 ms. Further, the neural representation of noun concreteness was modulated by the preceding adjectives: decoders learning concreteness signals in single words generalized better to subsective relative to privative phrases.

Vowel-like patterns modulate auditory P100m response but not its association with language abilities in children with ASD
Background: The P100/P100m component of auditory event-related potentials/fields is considered a potential biomarker of atypical arousal and language abnormalities in children with ASD. When elicited by complex speech-like sounds with regular temporal or frequency structure, P100/P100m may be influenced by sustained negativity (SN), which can reduce its amplitude due to opposing current polarity. Methods: Using MEG, we investigated how acoustic regularities affect P100m latency and amplitude differences between TD children and those with ASD. MEG was recorded in 35 ASD and 39 TD boys (7-12 years) in response to control sounds (non-periodic, non-vowels) and stimuli with temporal regularity (periodic non-vowels), frequency regularity (non-periodic vowels), or both (periodic vowels). P100m was estimated using distributed source localization. Results: In both groups, P100m amplitude and latency decreased in response to acoustic regularities, accompanied by a proportional increase in SN. No group differences were observed in P100m latency, amplitude, or their modulation by stimulus characteristics. In ASD, P100m latency variability was increased, and higher P100m amplitudes in the left auditory cortex were negatively associated with cumulative language and intellectual abilities. Conclusions: In children, changes in P100m in response to acoustic regularities are most parsimoniously explained by an enhancement of SN with opposite polarity. No consistent relationship was found between P100m parameters or their modulation by acoustic regularities and ASD diagnosis. However, variations in cortical maturation and/or habituation processes, which affect the left-hemispheric P100m, may be relevant to cognitive and language functioning in children with ASD.

Systematic modulation of sensorimotor learning by domain-specific working memory
People differ in how quickly they learn and adapt sensorimotor skills, and these differences have been linked to individual variation in working memory capacity (WMc). In tasks that can be supported by cognitive strategies, visuospatial WMc has been proposed as a key contributor. However, it remains unclear whether this association reflects domain-specific mechanisms or domain-general executive resources, and whether it extends beyond spatial memory to other visual features. Here, we systematically tested whether domain-specific or domain-general WMc predicts adaptation across three visuomotor adaptation (VMA) tasks known to differentially engage explicit and implicit learning systems. After obtaining independent measures of spatial and feature-based WMc, healthy subjects completed (1) a standard VMA task, which engages both explicit and implicit learning systems; (2) a delayed feedback VMA task, which isolates explicit learning; and (3) a clamped feedback VMA task, which isolates implicit learning. Our results provide converging evidence in support of a domain-specific association between spatial WMc and individual differences in learning. In Experiments 1 and 2, greater spatial WMc was associated with robust increases in explicit learning whereas featured-based WMc was not associated with learning outcomes. Surprisingly, in Experiment 3, greater spatial WMc was associated with reduced implicit learning, suggesting an interaction between latent cognitive capacities and implicit learning not accounted for by traditional models. These results shed light on the precise cognitive capacties underlying sensorimotor adaptation and provide novel insight into domain-specific links between spatial WM and motor learning.

Associations between symptom severity in Autism and functional neuroimaging measures of audiovisual speech perception.
Individuals on the Autism Spectrum do not benefit from visual articulatory cues when compared to neurotypicals especially under noisy environmental conditions. We hypothesized that this deficit would vary with the severity of Autism related symptoms and assessed this relationship in a behavioral speech in noise task (n = 32) and a functional neuroimaging study (n = 37). We found that Calibrated Symptom Severity Scores (CSS) were associated with poorer audiovisual performance but not performance in the auditory- alone condition indicating that impairments are limited to multisensory information processing. These findings underscore the validity of MS deficits and their potential relevance to the broader symptomatology in ASD. We also found that CSS significantly correlated with the hemodynamic responses to AV stimulation. Here, higher the symptom severity was associated with lower multisensory gain in dorsal speech and language regions. Subsequent exploratory analysis suggested that individuals with ASD may not engage speech motor regions in similar ways to TD individuals. These results differed from findings in our previous study (Ross et al., 2024) where a direct comparison between TD and ASD BOLD effect revealed differences in activation in mostly frontal regions, not associated with the task.

Reduced occurrence of alpha waves during resting state predicts high ADHD traits in young adults
Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental condition with significant cognitive and social impacts. Identifying reliable biomarkers for ADHD is crucial for developing personalized therapies. Electroencephalography (EEG) alpha oscillations (8 -12 Hz) have been suggested as a potential biomarker, but findings have been inconsistent. This study aimed to investigate whether alpha oscillations in young adulthood are associated with high ADHD traits using EEG data from a large twin sample (N = 556) enriched with participants with ADHD and autism. We assessed whether alpha oscillations during rest were associated with high ADHD traits through logistic regression analysis. In addition, we used twin modelling to estimate the heritability of EEG alpha measures and their relationship with ADHD traits. Results showed that relative alpha power was a significant predictor of ADHD traits when controlling for other factors such as age, sex and autistic traits. Specifically, we found that for each unit decrease in relative alpha power, the likelihood of being in the high ADHD trait group increased by approximately 26%. Further analysis suggested that group differences were due to a reduced occurrence (but not amplitude) of oscillatory bursts in the alpha range. Finally, our twin modelling results suggested that although alpha power is heritable, the genetic factors contributing to individual differences in alpha measures and ADHD traits were largely independent. Together, these findings suggest that reduced alpha oscillations, particularly occurrence of alpha bursts, may serve as a potential biomarker for ADHD. Our results may have implications for neuromodulation therapies targeting alpha rhythms in ADHD, such as neurofeedback and transcranial alternating current stimulation (tACS).