bioRxiv Subject Collection: Neuroscience's Journal

Multivesicular release at a small central excitatory synaptic contact
We set out to study the properties of excitatory synaptic transmission at a central connection likely to consist of a single small synaptic contact. In the cerebellar cortex, molecular layer interneurones extend sparse thin dendrites in a parasagittal plane. The nonbranching parallel fibres traverse at right angles to the dendritic plane, forming en passant excitatory synapses. This arrangement makes it very likely that any synaptic connection between a parallel fibre and an interneurone will involve only a single contact. We obtained 20 paired recordings of synaptically connected granule cells and interneurones in cerebellar slices of immature rat, recorded at room temperature. Three of these pairs were successfully labelled using biocytin and a single point of contact between them was indentified. In 19/20 recordings, multiple release events corresponding to asynchronously released vesicles were frequently resolved following a single action potential. The frequency of these events could not be accounted for by spontaneous EPSCs, whose frequency was very low. We conclude that at this excitatory synaptic contact an action potential can release more than one vesicle.

Left-to-right dorsomedial prefrontal cortex interhemispheric projections mediate psychosocial stress vulnerability
Functional asymmetries in the medial prefrontal cortex (mPFC) are significant attributes of this brain area, implicated in its role in emotional processing and executive function. Evidence suggests that, under normal conditions, there is a tonic inhibition of the right (R) mPFC by the left (L) mPFC, and a dysregulation of this hemispheric functional lateralization is implicated in detrimental chronic stress effects. Considering the wide interhemispheric connection and the inhibitory tone from the LmPFC to the RmPFC, we hypothesize that alterations in the activity of the direct projections between the mPFC hemispheres during stressful situations are related to stress vulnerability. To address this question, we used a chemogenetic approach to modulate the activity of L-to-R dorsomedial prefrontal cortex (dmPFC) monosynaptic projections during psychosocial stress (PSS) exposure in mice. We found that activating LdmPFC projections during a repeated PSS protocol prevents stress-induced apathy-like behavior in females and males and social avoidance in male mice. On the other hand, inhibiting such projections during a single session of PSS increases vulnerability to stress effects in male mice, increasing social avoidance and anxiety-like behaviors. Both glutamatergic and GABAergic cells compose the projecting interhemispheric neurons in the dmPFC. However, the LdmPFC showed a higher density of glutamatergic projections to the RdmPFC than the opposite. In conclusion, our results revealed an involvement of the monosynaptic projections from the LdmPFC to RdmPFC in the vulnerability to the behavioral alterations induced by PSS in female and male mice.

Cell-type-selective synaptogenesis during the development of excitatory connectivity in the mammalian neocortex
The function of mammalian neocortex relies on the timing of axon extension and establishment of cell-type-biased patterns of excitatory synaptic connections. A subtype of excitatory neurons, layer 6 corticothalamic neurons (L6CThNs), ultimately exhibit a marked preference for synapsing onto parvalbumin-positive (PV) inhibitory interneurons over more common excitatory cells in layers 6 and 4 (L6, L4). We show that the intracortical axons of L6CThNs develop in phases, elongating within L6, then pausing before extending translaminar branches into L4. Decreasing L6CThN excitability selectively enhanced axon growth in L6 but not later elaboration in L4. For both layers, we tested whether preferential synaptogenesis onto rarer PV interneurons, or promiscuous synapse formation followed by selective pruning, generated adult connectivity. We found that L6CThNs formed functional AMPA-receptor-containing synapses preferentially onto PV interneurons. Silent L6CThN synapses were not detected. Our findings show that cell-type-biased synaptogenesis underlies the formation of functional cell-type-specific excitatory connections in the neocortex.

Corticothalamic circuit mechanisms underlying brain region and ageing variations in resting-state alpha activity
Understanding the neural mechanisms underlying oscillations in resting-state brain activity, which exhibit substantial spatial and age-related variations, remains a significant challenge. This study aims to characterize the contributions of neural circuits to the mechanisms governing resting-state alpha oscillations, which are crucial for various neurocognitive processes and pathologies. Using the Cam-CAN dataset, source-space MEG analyses revealed a pronounced posterior-anterior gradient in alpha frequency, alpha power, and aperiodic components, alongside notable age-related changes. Through neurophysiological modelling, we uncover strong corticothalamic interactions in occipital regions, contrasting with predominantly corticocortical interactions in frontal areas. Ageing is associated with reduced intrathalamic activity and increased corticothalamic delay in occipital regions, while fronto-central regions exhibit increased intrathalamic activity. These findings establish how different circuits shape alpha oscillations across posterior-anterior axis and age, providing a mechanistic foundation for targeted clinical interventions and offering benchmarks for future studies in patient populations.

Wide-ranging behavioral dysfunction in two mouse models of pathological human variants in the GRIK2 kainate receptor gene
De novo variants in a subset of ionotropic glutamate receptor (iGluR) genes cause nonsyndromic neurodevelopmental disorders in individuals. Two recurrent variants in the kainate receptor (KAR) gene GRIK2 result in the gain-of-function (GoF) substitutions p.Ala657Thr and p.Thr660Lys in a critical pore-forming domain of the GluK2 subunit. Disorders in individuals with these variants manifest as intellectual disability, developmental delay, motor impairments, and, in the case of p.Thr660Lys, epilepsy. To explore their pathogenicity and phenotypic consequences in vivo, we generated knock-in mouse models harboring orthologous Grik2 mutations. Behavioral analyses revealed a range of developmental, motor, cognitive, and naturalistic behavior impairments in both lines, with T660K mice typically exhibiting more severe phenotypes, consistent with clinical observations in humans. GluK2(T660K) mice also display interictal EEG abnormalities and handling-induced seizures. These models establish the first in vivo platforms for dissecting the underlying mechanisms of NDDs caused by a GoF mutation in the GluK2 KAR subunit and represent crucial tools for therapeutic development.

N100 as a Neural Marker of Atypical Early Auditory Encoding in Autism: Sensitivity to Pitch, Distance-Based Intensity, and Spatial Location
Background: Individuals with Autism Spectrum Disorder (ASD) show atypical auditory perception. The N100 event-related potential (ERP) reflects early auditory encoding, predictive coding, and sensory gain. Therefore, this study examined N100 responses to speech stimuli as a neural marker of auditory processing differences in ASD. Methods: Event related potentials (ERPs) were recorded using OpenBCI in 12 boys diagnosed with Level 1 ASD (requiring minimal support) and 15 typically developing (TD) peers. Participants passively listened to Romanian sentences systematically varied in pitch (normal, high, low), distance-based intensity (0.5, 1, 2 meters; 65, 59, 53 dB), and spatial presentation (binaural, left, right). N100 amplitudes and latencies were analyzed using Python and SPSS. Results: ASD group indicated significantly reduced N100 amplitudes for normal pitch stimuli (p = .030, = .175) and binaural presentation (p = .030, = .175). Marginal reductions were also observed for low pitch (p = .096, = .120), speech presented from a 0.5-meter distance (p = .058, = .147), and unilateral conditions (ps = .066.077, s = .130.142). No group differences emerged for N100 latency. These findings suggest attenuated early auditory responses in ASD to both typical and spatially complex speech cues. Conclusions: Results support predictive coding models proposing reduced sensory precision in ASD. The consistent amplitude attenuation, including near-significant findings, points to subtle but pervasive impairments in early auditory encoding. The use of ecologically valid speech stimuli and portable EEG underscores the translational potential of N100 as a biomarker for early identification and intervention in autism.

From one schema to another: How the prefrontal cortex responds to conflicting information
Over time, we develop event schemas or scripts that shape our expectations about what typically happens in certain contexts. However, even after forming a memory about a certain event, we are often exposed to related information about that same event at later points in time. This additional information sometimes causes one to have to re-evaluate the interpretation of the original event. Over a two-day fNIRS experiment, participants were exposed to events that were subsequently updated with schema-congruent or schema-incongruent additional details. These schema-incongruent additional details make those events more fitting to another schema than originally was the case, meaning that participants would need to dissociate that event from the original schema and re-integrate it with another schema. The fNIRS results showed stronger PFC activity for events updated with schema-congruent compared to schema-incongruent details. When specifically looking at those events that were updated with schema-incongruent details, our results suggest that dissociating an event from the original schema and re-integrating it with another schema was accompanied by an initial PFC decrease early in the trial followed by a PFC increase later in the trial. This was a distinctly different pattern compared to trials in which participants failed to re-integrate the event with another schema, which showed delayed PFC increase with lower amplitude and no initial PFC decrease. Our results refine our understanding of mechanisms of adaptive memory updating in the face of conflicting information.

A Core Pattern of Cerebellar and Brainstem Degeneration and Reduced Cerebrocerebellar Structural Covariance in Spinocerebellar Ataxia Type 3 (SCA3): MRI Volumetrics from ENIGMA-Ataxia
Objective: Spinocerebellar ataxia type 3 (SCA3) is a rare, inherited neurodegenerative disease. Here, we profile the spatial spread of atrophy across the whole brain, determine whether brain degeneration preferentially maps onto specific functional networks, and investigate the relationship between cerebellar and cerebral anatomical changes. Methods: Whole-brain grey and white matter (GM and WM) voxel-based morphometry was performed on 408 individuals with SCA3 (82 pre-ataxic) and 293 controls. The SCA3 cohort was stratified by ataxia severity to investigate disease progression, with cerebellar GM atrophy mapped onto a task-based functional atlas. Volume was correlated with disease duration and intensity. Cerebrocerebellar volumetric covariance was assessed to determine whether atrophy was coupled between infra- and supratentorial regions. Results: The pattern of atrophy is spatially consistent but progressive in magnitude across the disease course. The greatest atrophy was found in the pons, cerebellar WM, and cerebellar peduncles; correlations with disease severity and duration were also strongest in these regions. Cerebellar GM atrophy was greatest in functional regions associated with motor execution and planning, attention, and emotional processing. Sparse cerebral cortical atrophy appears only in the most severe disease subgroup, while striatal atrophy begins in the earliest stages. Reduced cerebrocerebellar structural covariance is observed in SCA3 participants versus controls. Interpretation: While cerebellar and brainstem atrophy become more severe, the pattern of atrophy remains largely consistent as SCA3 progresses. Cerebellar GM degeneration occurs in regions associated with motor, cognitive, and affective control, in line with clinical presentation. Cerebellar atrophy is not directly mirrored by cerebral changes.

Two forms of uncertainty hierarchically shape lexical predictions during natural speech processing
Speech comprehension relies on contextual lexical predictions to optimize the processing of incoming words. As in other predictive paradigms, it is commonly assumed that prediction error, or surprisal, is weighted by a single summary statistic: uncertainty. Uncertainty reflects the overall unpredictability of the context and is typically quantified using Shannon entropy. However, for predictions involving many alternatives, as in speech, Shannon entropy is an ambiguous measure. We investigated alternative measures of uncertainty, namely the family of Renyi entropies, of which Shannon entropy is a special case. Using whole-brain intracranial EEG recordings in 33 epilepsy patients listening to natural, continuous speech, we show that, rather than Shannon entropy, contextual lexical uncertainty in speech is best captured by two extremes of the Renyi family. These reflect the probability of the most likely word and the spread across all plausible alternatives, which we term strength and dispersion, respectively. These representations are processed in distinct neural populations in a spatiotemporal hierarchy, with the encoding of dispersion preceding that of strength. Around each word onset, information flows between these clusters in a top-down and subsequently bottom-up sequence. Finally, they interact differentially with word surprisal during word processing. Our findings demonstrate that the brain encodes a multidimensional representation of uncertainty and reveal that multiple summary statistics may coexist in predictive processing. More broadly, this work introduces a flexible framework for characterizing the neural bases of uncertainty. It also highlights the potential mismatch between formal definitions of cognitive variables and their neural implementation.

Inhibiting the right dorsolateral prefrontal cortex selectively enhances unsupervised statistical learning
The brain must balance the automatic extraction of environmental regularities with top-down cognitive control, yet the causal neural mechanisms governing this interplay are debated. In particular, the hemispheric contributions of the dorsolateral prefrontal cortex (DLPFC) remain unresolved. Here, we applied inhibitory repetitive transcranial magnetic stimulation (rTMS) to the left, right, or bilateral DLPFC in 95 healthy adults during a probabilistic sequence learning task. We found that inhibiting the right and bilateral DLPFC significantly enhanced statistical learning compared to sham and left DLPFC stimulation. Contrary to the hypothesis that this competition is mediated by the suppression of existing knowledge, episodic memory performance was unaffected across all stimulation groups. These findings challenge a direct episodic gating mechanism and suggest the DLPFC's influence on statistical learning is hemisphere-dependent. As an alternative, we propose that right DLPFC inhibition shifts cognitive processing toward a more exploratory information-sampling strategy, a view supported by our finding of significantly greater reaction time variability in the right and bilateral stimulation groups. Together, our results provide causal evidence for a right-lateralized suppressive influence of the DLPFC on unsupervised learning and suggest its modulatory role is linked to information processing style rather than direct competition with episodic memory systems.

Autophagy Dysfunction in iPSCs-Derived Neurons and Midbrain Organoids Carrying a SNCA Triplication
Parkinsons disease (PD), characterized by a-Synuclein aggregation and dopaminergic neuronal loss, has no current cure. Autophagy is critical for a-Synuclein clearance, yet its real-time dynamics remain challenging to assess in human-relevant systems. Here, we used live-cell imaging to assess autophagy within human neuronal cultures and midbrain organoids (hMOs) derived from induced pluripotent stem cells (iPSCs) of PD patients carrying a triplication of the a-Synuclein gene (3xSNCA). Using the LC3-Rosella dual-fluorescent reporter, we quantified autolysosomes dynamics in real time. In 3xSNCA neuronal cultures, we detected early autophagy defects. In 3xSNCA hMOs, reduced autolysosome area, increased total and phosphorylated a-Synuclein (pS129), and decreased electrophysiological activity were observed at 50 days of differentiation (DoD). By 70 DoD, autophagy impairment became more pronounced, overlapping with dopaminergic neuron loss. These findings support the use of human iPSCs-derived models to study autophagy dysfunction in PD and demonstrate a temporal correlation between impaired autophagy, a-Synuclein pathology and neuronal degeneration.

Visuospatial working memory is cortically enabled through veridical, categorical and semantic representations
A single visual stimulus can elicit multiple concurrent representations throughout the cortex. We show that during a visual working memory task several cortical regions utilize categorical, semantic, and spatial representational formats to maintain visual stimuli in a robust fashion. We assessed the nature of orientation representations in an fMRI dataset of 40 participants performing an orientation memory task using multivariate encoding modelling. Our results show that orientation representations across the cortex form a gradient of abstraction, with more veridical representations in sensory areas and more abstract, categorical codes in anterior areas. We use cross-condition modelling to demonstrate shared neural codes between orientation and congruent verbal and location stimuli, used at different points during the trial. These findings provide evidence for a distributed account of working memory storage, where memory representations, distinct in nature and content, are present concurrently throughout the cortex.

Localization of Mutant Huntingtin with HTT Exon1 P90 C-terminal Neoepitope Antibodies in Relation to Regional and Neuronal Vulnerability in Forebrain in Q175 Mice and Human Huntington's Disease
Background Recent evidence suggests that accumulation of mutant exon 1 protein may be critical to HD pathogenesis, but the relation of this to differential regional and cellular vulnerability in HD is unknown. Objective We assessed the contribution of the accumulation of the mutant huntingtin exon 1 protein to the regional and cellular variation in HD brain pathology by determining if more vulnerable regions and neuron types were relatively enriched. Methods We performed immunolabeling using the novel monoclonal antibodies 11G2 and 1B12 against the C-terminal proline 90 (P90) neoepitope of the huntingtin exon 1 protein on forebrain of Q175 mice and human HD cases, which detect accumulation of monomeric, oligomeric and aggregates mutant exon 1 protein. Results Diffuse nuclear and aggregate immunolabeling increased in abundance in Q175 with age, with striatal projection neurons showing immunolabeling earlier than cortical neurons, and pallidal regions only showing neuropil immunolabeling. Nonetheless, some regions less affected in HD, such as hippocampus, were rich in mutant exon 1 protein as well. In humans, striatal immunolabeling was sparser than in mouse, and mainly in the neuropil, but less sparse than in striatal target areas. In human HD cortex, the P90 antibodies detected predominantly neuropil aggregates, which appeared to, in part, localize to dendrites. Conclusions Our results indicate that exon 1 mutant protein burden does appear to partly account for overall forebrain vulnerability in HD, but additional factors may contribute to vulnerability differences among forebrain regions and between specific neuron types.