Biology subjects

Heintz, N.

Publications and source records attributed to Heintz, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Reconstructing the developmental history of the cortex from postmortem tissue

Single-cell-resolution mapping of the human brain is central to understanding the link between cellular-level phenotypes and disease. Tissue clearing and mesoscale imaging have facilitated organ-wide quantification of cell populations and are expected to drive the identification of pathological deficits in neurological disorders. However, mesoscopic comparison of human brain tissue remains challenging because of large variability in cortical folding patterns and heterogeneous cell distribution. Here, we present a "retrodictive" analysis of the cortex that reconstructs the proliferation history of neural stem cells from a static snapshot of tissue, providing a developmentally grounded coordinate system for comparison across samples. The analysis showed that most of the heterogeneity in cell distribution can be explained by the physical displacement caused by folding, which allows folding-induced variation to be removed and additionally reveals a possible mechanism of cortical folding. Together with a Bayesian framework, we estimated, with quantified uncertainty, the proliferation rate of a representative stem cell that summarizes the population of each cortical region. This analysis enables researchers to distill inter-individual differences down to stem cell properties and their distributions on the protomap. We anticipate our workflow to be a foundational framework for 3D neuropathological analysis, providing a common coordinate for the cortex-wide analysis of cells across individuals in development, aging, and disease.

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bioRxiv2026

When feedback backfires: investigating neurofeedback effects in a closed-loop auditory attention decoding paradigm

Selective auditory attention decoding (AAD) enables tracking which of multiple concurrent speakers a listener attends to and is a key building block for neuro-steered hearing devices. While AAD integrated in a closed-loop system with real-time neurofeedback (NFB) is hypothesized to improve decoding through neural adaptation and error-correction behaviour, the short-term behavioral and algorithmic impact of such a bilateral human-machine interaction remains poorly understood. Here we evaluated the effects of NFB on AAD accuracy and user experience in a single-session AAD paradigm with online NFB involving nineteen participants. They performed a selective listening task with enforced attention switches across four conditions: open-loop (OL), closed-loop with auditory gain feedback (CLA), closed-loop with visual feedback (CLV), and a condition with pseudo-auditory gain control (psCLA) decoupled from the participants individual neural activity. AAD was performed online using both subject-specific and subject-independent linear decoders on 5 s sliding windows, followed by Hidden Markov Model post-processing. Online analysis showed comparable decoding performance across all conditions. However, offline posthoc analysis using subject-independent decoders revealed that AAD accuracy in the CLA condition was significantly lower than in the OL baseline. Subjectively, participants reported that CLA was significantly more distracting and required higher switching effort. Crucially, a causal analysis of the psCLA condition found no robust evidence that higher audio gains inherently improve decoding accuracy. Our results demonstrate that within a single-session paradigm with rapidly varying feedback cues, auditory neurofeedback may degrade AAD performance by increasing cognitive load and distraction. These findings suggest that suboptimal feedback can impede rather than facilitate learning. We conclude that more accurate and stable decoders and longitudinal, multi-session training protocols are likely essential prerequisites for achieving beneficial neurofeedback effects in closed-loop auditory attention systems.

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bioRxiv2026

GPCRs as Targets for Human Brain Modulation: A Multi-omic Atlas of Cell-Type Specific Expression

G Protein coupled receptors (GPCRs) are the largest class of clinically validated drug targets with nearly 35% of all approved therapeutic agents acting on these receptors. To further explore the potential of this class of receptors for the development of circuit-specific and mechanism-based therapeutic strategies for neurological disorders, we focused on GPCRs with no known endogenous ligand, orphan GPCRs (oGPCRs), because knowledge of their functions in the human brain remains rudimentary. Here, we utilized fluorescence activated nuclear sorting and sequencing (FANSseq) to generate deep molecular profiles of cell type specific nuclei isolated from post-mortem brains to generate an atlas of oGPCR expression across multiple regions of the human brain. We identified 22 oGPCRs that displayed selective cell-type enrichment both in RNA transcript expression and chromatin accessibility. We further validated each of these targets for cell-type specific expression in human brains and developed an open-source web atlas of all oGPCR expression in the human brain to serve as a neuro-resource for the broader scientific community. These studies reveal novel cell-type specific expression patterns of several oGPCRs, suggest potential endogenous roles for these receptors, and identify validated candidates for cell-type specific neuromodulation of the human brain. One Sentence SummaryThis study presents an atlas of orphan GPCR expression across the human brain for translational targeting.

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bioRxiv2026

Loss of interruption in the HTT CAG repeat is associated with increased somatic expansion and loss of medium spiny neurons in HD

Synonymous loss of interruption variants in the expanded CAG repeat sequence of Huntingtin (HTT) accelerate the clinical onset and progression of Huntington disease (HD). Medium spiny neurons (MSNs) are gradually lost in HD and undergo selective somatic CAG expansion, but it is unclear how CAG expansion in MSNs relates to HD pathology. Here, we show that MSNs with large (111-150 CAG) and very large (>150 CAG) somatic expansions are rare in early manifest HD, but accumulate in proportion with duration of disease and inherited CAG repeat length. In patients with the deleterious CAG-CCG loss-of-interruption (CAG-CCG LOI) modifier, the proportion of MSNs with large and very large expansions is increased [~]5-fold despite reduced small somatic expansions in blood, and direct caudate MSN counts are reduced. Our findings suggest that increased somatic CAG expansion contributes to accelerated striatal MSN pathology and hastened onset of HD, but that MSNs with very large genomic CAG expansions can persist among surviving neurons of the HD brain.

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bioRxiv2026

Divergent mitochondrial and metabolic adaptations shape selective vulnerability in ALS

Neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) exhibit striking cell-type selectivity, yet the basis for this vulnerability remains elusive. Here, we uncover that even closely related neurons can harbor distinct mitochondrial properties that shape their response to disease. Using TOM-Tag, a circuit-based AAV-based strategy for cell type-specific mitochondrial immunopurification from projection neurons, we performed integrative proteomic, metabolomic, transcriptomic, and functional analyses of mitochondria from ALS-vulnerable corticospinal projection neurons (CSPNs) and resilient corticothalamic projection neurons (CTPNs) in vivo. We discovered that CSPNs and CTPNs exhibit divergent mitochondrial profiles at baseline, despite sharing cortical layer and developmental origin. CTPNs were primed for antioxidant buffering and fatty acid metabolism, whereas CSPNs were enriched for oxidative phosphorylation components. In ALS, CTPNs employed mitochondrial flexibility and redox defense, whereas CSPNs exhibited respiratory failure and metabolic stress. These findings reveal that intrinsic mitochondrial programs vary even between similar neurons, and that this hidden layer of diversity may critically shape susceptibility to neurodegeneration. By enabling high-resolution access to mitochondria in defined neuronal circuits, TOM-Tag offers a powerful new lens for dissecting disease mechanisms and identifying cell-specific therapeutic targets.

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bioRxiv2025

Artificial Intelligence-driven Whole-brain Cell Mapping with Highly Multiplexed In Situ Hybridization

Recent advances in three-dimensional single-cell-resolution imaging have begun to link organ-wide and cellular level research in development and disease research. Harnessing the power of whole-mount cell staining and tissue-clearing, it became possible to quantify the cell populations throughout an intact organ. While powerful, whole-organ imaging remains limited by the inability to stain a broad range of molecular markers simultaneously and by the lack of an analytical scheme to precisely quantify the cell population. Here, we present a highly multiplexed whole-mount staining technique, utilizing the repeated application of fluorescent in situ hybridization. This technique, termed mFISH3D, was designed by extensively dissecting the chemical basis of hybridization reactions in fixed tissue. mFISH3D enabled the visualization of 10 types of mRNAs in an intact mouse brain and has been demonstrated in various biological specimens including the human brain. To achieve unprecedented levels of accuracy in spatial cell mapping, we developed artificial intelligence (AI)-driven workflow using self-supervised learning, significantly reducing the need for extensive manual annotations. The integration of mFISH3D with our AI solution sets a standard for high-dimensional tissue analysis, provides a new systematic framework for analyzing complex cellular ecosystems and enables comprehensive investigation of selective cellular vulnerabilities in diseases.

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bioRxiv2025

EEG-based Decoding of Auditory Attention to Conversations with Turn-taking Speakers

ObjectivesAuditory attention decoding (AAD) refers to the process of identifying which sound source a listener is attending to, based on neural recordings, such as electroencephalography (EEG). Most AAD studies use a competing speaker paradigm where two continuously active speech signals are simultaneously presented, in which the participant is instructed to attend to one speaker and ignore the other speaker. However, such a competing two-speaker scenario is uncommon in real life, as speakers typically take turns rather than speaking simultaneously. In this paper, we argue that decoding attention to conversations (rather than individual speakers) is a more relevant paradigm for testing AAD algorithms. In such a conversation-tracking paradigm, the AAD algorithm focusses on switching between entire conversations, resulting in less frequent attention shifts (ignoring turn-taking within conversations), thereby allowing for more relaxed constraints on the decision time. DesignTo test AAD performance in such a conversation-tracking paradigm, we simulated a challenging restaurant scenario with three simultaneous two-speaker conversations, which were podcasts presented in front of the listener and in the back left and back right of the room. We conducted an EEG experiment on 20 normal-hearing participants to compare the performance of AAD in the commonly used competing speaker paradigm with two speakers versus the conversation tracking paradigm with 2 or 3 conversations, each containing two turn-taking speakers. ResultsWe found that AAD, using stimulus decoding, worked well under all experimental conditions, and that the accuracy was not influenced by the direction of attention, the proximity to the target conversation, or the presence of within-trial attention switches (versus a condition with sustained attention). Given the challenging scenario, we probed for the participants listening experience and found a correlation between the neural decoding performance and the perceived listening effort and self-reported speech intelligibility. To gain insight into the speech intelligibility of the participants in our setup, they performed a speech-in-noise test (Flemish matrix sentence test), but we did not find a correlation between the speech intelligibility performance and the AAD performance.

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bioRxiv2025

Effects of speaker and listener sex on auditory attention decoding performance

ObjectivesAuditory Attention Decoding (AAD) is a technique utilizing brain signals to decode on which sound the listener focuses the attention. In most current studies, the effect of type of speech materials used and sex of the listener is not considered. We investigated the effect on AAD performance of factors related to the speaker (such as the sex of the speaker, background noise level, and same versus mixed-sex conditions) and the listener (sex of the listener). DesignForty-two young adults with normal hearing participated in the study. They listened to 2 competing speakers and were instructed to attend to one speaker and ignore the other speaker, whilst electroencephalography (EEG) and electrooculography (EOG) were measured. Background noise was introduced in half of the conditions. AAD performance was compared across eight experimental conditions. ResultsA significant main effect of speaker sex was found: A male target and/or male masker speaker resulted in higher AAD performance compared to a female speaker with a higher fundamental frequency. These effects were found to be small and therefore likely clinically irrelevant. ConclusionWhile no substantial effects were found on the factors investigated in this study, including diverse and realistic training scenarios remains a valuable approach to prevent potential influences from other factors.

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bioRxiv2025

Epigenetic mechanisms governing cell type specific somatic expansion and toxicity in Huntington's disease

Huntingtons disease (HD) is characterized by neuronal dysfunction and degeneration that varies markedly by brain region and cell type. Using high-resolution epigenetic profiling of postmortem human cell types we identify a pathogenic cascade linking cell type specific enhancer activity to somatic CAG expansion, and toxicity to epigenetic dysregulation. Enhancers regulating mismatch-repair (MMR) gene expression explain the specificity of expansion. In the second, toxic phase of HD we identify two distinct epigenetic mechanisms that disrupt regulation of hundreds of genes in the majority of HD MSNs, including several that cause haploinsufficient neurological disorders. Together, these data unify enhancer function, impaired DNA demethylation, and transcriptional dysregulation into a single model highlighting therapeutic opportunities that combine inhibition of somatic CAG expansion with restoration of neuronal DNA demethylation.

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bioRxiv2025

Identification of a Thermogenic Target in the Dorsal Raphe Nucleus for Weight Management

Obesity emerges from a complex interplay of factors, including imbalanced interoception, genetic predisposition, and environmental cues, ultimately disrupting body weight homeostasis1. While much research has concentrated on strategies to suppress appetite for sustained weight loss, insufficient attention has been given to counterregulatory mechanisms that promote energy expenditure. Here, we show that chronic inhibition of GABAergic neurons in the Dorsal Raphe Nucleus (DRNVGAT) reduces body weight in diet-induced obese (DIO) mice. In this study, molecular profiling and in-situ hybridization in rodent and human brains revealed that the constitutively activated orphan receptor GPR6 is selectively enriched in DRNVGAT neurons. We next developed and administered a potent and highly selective GPR6 inverse agonist, which significantly reduced weight gain in DIO mice by stimulating brown adipose tissue thermogenesis without affecting appetite. Altogether, this study transitions from transcriptomic profiling, high-throughput drug screening and metabolic phenotyping to successfully identify a novel candidate to treat obesity.

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bioRxiv2025

Open-source Photobleacher for Fluorescent Imaging of Large Pigment-Rich Tissues

Fluorescent imaging enables visualization of the specific molecules of interest with high contrast, and the use of multiple fluorophores in a single tissue sample allows visualization of complex relationships between biological molecules, cell types, and anatomy. The utility of fluorescent imaging in human tissue has been limited by endogenous pigments that can block the light path or emit an autofluorescence, thereby interfering with the specific imaging of target molecules. Although photobleachers have been developed to quench endogenous pigments, the lack of customizability limits their utility for a broad range of applications. Here, we present a high luminous-intensity photobleacher that is based on rigorous simulations of illumination patterns using the laws of radiation, along with the framework to maximize bleaching efficiency. This open-source project is designed to help researchers customize and scale according to the tissue types and the research goals. The photobleacher is applicable to both thin tissue slices and large-volume cleared tissue samples to enable serial three-dimensional imaging of postmortem human brain using multiplexed antibody or oligonucleotide probes. SIGNIFICANCE STATEMENTPhotobleaching is an effective technique for quenching endogenous pigments, enabling multiplexed fluorescent imaging of pigment-rich tissues, such as postmortem human samples. While many photobleaching strategies have been proposed, there is no standard guidance on how to design and use a photobleacher. This study introduces a general strategy for designing an effective, scalable, and customizable photobleacher, and proposes a workflow for properly treating tissues with the photobleacher. The technique enables high-contrast molecular visualization in tissues of various sizes, including large volumetric cleared tissues. Our framework will accelerate the quantitative understanding of human molecular anatomy and is applicable to diverse biological fields, including medical diagnostics.

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bioRxiv2024

Spontaneously regenerative corticospinal neurons in mice

The spinal cord receives inputs from the cortex via corticospinal neurons (CSNs). While predominantly a contralateral projection, a less-investigated minority of its axons terminate in the ipsilateral spinal cord. We analyzed the spatial and molecular properties of these ipsilateral axons and their post-synaptic targets in mice and found they project primarily to the ventral horn, including directly to motor neurons. Barcode-based reconstruction of the ipsilateral axons revealed a class of primarily bilaterally-projecting CSNs with a distinct cortical distribution. The molecular properties of these ipsilaterally-projecting CSNs (IP-CSNs) are strikingly similar to the previously described molecular signature of embryonic-like regenerating CSNs. Finally, we show that IP-CSNs are spontaneously regenerative after spinal cord injury. The discovery of a class of spontaneously regenerative CSNs may prove valuable to the study of spinal cord injury. Additionally, this work suggests that the retention of juvenile-like characteristics may be a widespread phenomenon in adult nervous systems.

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bioRxiv2024

Estrus-Tracking Cortical Neurons Integrate Social cues and Reproductive states to Adaptively Control Sexually Dimorphic Sociosexual Behaviors

Female sociosexual behaviors, essential for survival and reproduction, are adaptively modulated by ovarian hormones. However, the neural mechanisms integrating internal hormonal states with external social cues to guide these behaviors remain poorly understood. Here we identified primary estrous-sensitive Cacna1h-expressing medial prefrontal (mPFCCacna1h+) neurons that orchestrate adaptive sociosexual behaviors. Bidirectional manipulation of mPFCCacna1h+ neurons drives opposite-sex-directed behavioral shifts between estrus and diestrus females. In males, these neurons serve opposite functions compared to estrus females, mediating sexually dimorphic effects via anterior hypothalamic outputs. Miniscope imaging reveals mixed-representation of self-estrous states and social target sex in distinct mPFCCacna1h+ subpopulations, with biased-encoding of opposite-sex social cues in estrus females and males. Mechanistically, ovarian hormone-driven upregulation of Cacna1h-encoded T-type calcium channels underlies estrus-specific activity changes and sexual-dimorphic function of mPFCCacna1h+ neurons. These findings uncover a prefrontal circuit that integrates internal hormonal states and target-sex information to exert sexually bivalent top-down control over adaptive social behaviors.

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bioRxiv2024

Opto-CLIP reveals dynamic FMRP regulation of mRNAs upon CA1 neuronal activation

Neuronal diversity and function are intricately linked to the dynamic regulation of RNA metabolism. Electrophysiologic studies of synaptic plasticity, models for learning and memory, are disrupted in Fragile X Syndrome (FXS). FXS is characterized by the loss of FMRP, an RNA-binding protein (RBP) known to suppress translation of specific neuronal RNAs. Synaptic plasticity in CA1 excitatory hippocampal neurons is protein-synthesis dependent, suggesting a role for FMRP in FXS-related synaptic deficits. To explore this model, we developed Opto-CLIP, integrating optogenetics with cell-type specific FMRP-CLIP and RiboTag in CA1 neurons, allowing investigation of activity-induced FMRP regulation. We tracked changes in FMRP binding and ribosome-associated RNA profiles 30 minutes after neuronal activation. Our findings reveal distinct temporal dynamics for FMRP transcript regulation in the cell body versus the synapse. In the cell body, FMRP binding to transcripts encoding nuclear functions is relieved, potentially allowing rapid transcriptional responses to neuronal activation. At the synapse, FMRP binding to transcripts encoding synaptic targets was relatively stable, with variability in translational control across target categories. These results offer fresh insights into the dynamic regulation of RNA by FMRP in response to neuronal activation and provide a foundation for future research into the mechanisms of RBP-mediated synaptic plasticity.

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bioRxiv2023

'Are you even listening?' - EEG-based detection of absolute auditory attention to natural speech with application to neuro-steered hearing devices

ObjectiveIn this study, we use electroencephalography (EEG) recordings to determine whether a subject is actively listening to a presented speech stimulus. More precisely, we aim to discriminate between an active listening condition, and a distractor condition where subjects focus on an unrelated distractor task while being exposed to a speech stimulus. We refer to this task as absolute auditory attention decoding. ApproachWe re-use an existing EEG dataset where the subjects watch a silent movie as a distractor condition, and introduce a new dataset with two distractor conditions (silently reading a text and performing arithmetic exercises). We focus on two EEG features, namely neural envelope tracking (NET) and spectral entropy (SE). Additionally, we investigate whether the detection of such an active listening condition can be combined with a selective auditory attention decoding task, where the goal is to decide to which of multiple competing speakers the subject is attending. The latter is a key task in so-called neuro-steered hearing devices that aim to suppress unattended audio, while preserving the attended speaker. Main resultsContrary to a previous hypothesis of higher SE being related with actively listening rather than passively listening (without any distractors), we find significantly lower SE in the active listening condition compared to the distractor conditions. Nevertheless, the NET is consistently significantly higher when actively listening. Similarly, we show that the accuracy of a selective auditory attention decoding task improves when evaluating the accuracy only on the highest NET segments. However, the reverse is observed when evaluating the accuracy only on the lowest SE segments. SignificanceWe conclude that the NET is more reliable for decoding absolute auditory attention as it is consistently higher when actively listening, whereas the relation of the SE between actively and passively listening seems to depend on the nature of the distractor.

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bioRxiv2023

EEG-based decoding of the spatial focus of auditory attention in a multi-talker audiovisual experiment using Common Spatial Patterns

ObjectiveSpatial auditory attention decoding (Sp-AAD) refers to the task of identifying the direction of the speaker to which a person is attending in a multi-talker setting, based on the listeners neural recordings, e.g., electroencephalography (EEG). The goal of this study is to thoroughly investigate potential biases when training such Sp-AAD decoders on EEG data, particularly eye-gaze biases and latent trial-dependent confounds, which may result in Sp-AAD models that decode eye-gaze or trial-specific fingerprints rather than spatial auditory attention. ApproachWe designed a two-speaker audiovisual Sp-AAD protocol in which the spatial auditory and visual attention were enforced to be either congruent or incongruent, and we recorded EEG data from sixteen participants undergoing several trials recorded at distinct timepoints. We trained a simple linear model for Sp-AAD based on common spatial patterns (CSP) filters in combination with either linear discriminant analysis (LDA) or k-means clustering, and evaluated them both across- and within-trial. Main resultsWe found that even a simple linear Sp-AAD model is susceptible to overfitting to confounding signal patterns such as eye-gaze and trial fingerprints (e.g., due to feature shifts across trials), resulting in artificially high decoding accuracies. Furthermore, we found that changes in the EEG signal statistics across trials deteriorate the trial generalization of the classifier, even when the latter is retrained on the test trial with an unsupervised algorithm. SignificanceCollectively, our findings confirm that there exist subtle biases and confounds that can strongly interfere with the decoding of spatial auditory attention from EEG. It is expected that more complicated non-linear models based on deep neural networks, which are often used for Sp-AAD, are even more vulnerable to such biases. Future work should perform experiments and model evaluations that avoid and/or control for such biases in Sp-AAD tasks.

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bioRxiv2023

Cell Type Specific CAG Repeat Expansion and Toxicity of Mutant Huntingtin in Human Striatal and Cerebellar Cell Types

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntingtons disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutS{beta}), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

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bioRxiv2023

Layer 5a Corticostriatal Projection Neurons are Selectively Vulnerable in Huntington's Disease

The properties of the cell types that are selectively vulnerable in Huntingtons disease (HD) cortex, the nature of somatic CAG expansions of mHTT in these cells, and their importance in CNS circuitry have not been delineated. Here we employed serial fluorescence activated nuclear sorting (sFANS), deep molecular profiling, and single nucleus RNA sequencing (snRNAseq) to demonstrate that layer 5a pyramidal neurons are vulnerable in primary motor cortex and other cortical areas of HD donors. Extensive mHTT-CAG expansions occur in vulnerable layer 5a pyramidal cells, and in Betz cells, layer 6a, layer 6b neurons that are resilient in HD. Retrograde tracing experiments in macaque brains identify the vulnerable layer 5a neurons as corticostriatal pyramidal cells. We propose that enhanced somatic mHTT-CAG expansion and altered synaptic function act together to cause corticostriatal disconnection and selective neuronal vulnerability in the HD cerebral cortex.

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