Biology subjects

Stauffer, W. R.

Publications and source records attributed to Stauffer, W. R..

15 recordsLinked to original sources

bioRxiv2026

Fine-mapping candidate neuropsychiatric regulatory variants using cell type-aware comparative genomics

AbstractMeasures of nucleotide sequence conservation across species are useful for identifying functional genomic loci, but can fail when regulatory function is maintained, often in a cell type-specific manner, even when sequence is not. We introduce CTACIT, the Cell Type-Aware Conservation Inference Toolkit, to identify trait-associated regulatory variants. CTACIT integrates sequence conservation scores with cell type-specific open chromatin data collected from a few mammalian species to impute function for hundreds more. Applying CTACIT to neuropsychiatric trait loci identifies higher heritability enrichment and more fine-mapped variants than nucleotide conservation and human chromatin data alone. Our in vivo reporter assays validate predictions for enhancers with risk variants near the DRD2 schizophrenia risk locus. By integrating genome conservation and multi-species open chromatin data, CTACIT prioritizes variants within regions of conserved regulatory function for in vivo characterization and addresses a major challenge in translating disease associations to mechanistic understanding. One-Sentence SummaryRegulatory functions conserved across mammals underlying caudate cell type evolution reveal functions of human risk variants.

genomics↗

bioRxiv2026

Structural dynamics insights into principles underlying the fitness of new broadly potent AAVs

Adeno-associated virus (AAV) is a leading platform for gene therapy, but current clinical-stage vectors require high doses associated with adverse events. Engineering of AAVs has produced more efficient vectors, although the mechanism underlying these improvements often remains poorly understood, limiting further development and raising potential safety concerns. Here, we leveraged a new workflow for AAV engineering with single-cell resolution, called scAAVengr-Hunt, to create best-in-class AAVs for gene delivery. ATX002, the top-performing vector, demonstrates broad potency across species, including nonhuman primate, mouse, and human, as well as across retina and brain. To understand the mechanism underlying this broad potency, we performed molecular dynamics simulations comparing AAV variants spanning a range of fitness levels. Structural dynamics analysis revealed a bifunctional molecular mechanism that confers potency through increased affinity of the capsid to the AAV receptor and regulation of heparan sulfate binding. This work provides critical insights relating structural mechanism to the fitness of engineered AAVs and establishes rich new avenues for AAV engineering through the integration of sequence-level analysis with computational biophysics.

bioengineering↗

bioRxiv2026

Neural correlates of combinatorial reasoning in prefrontal cortex

Complex economic decisions are often combinatorial: they require individuals to select from many alternatives under strict constraints on time, resources, and energy. Combinatorial reasoning is the cognitive process that enables decision makers to construct and evaluate multiple potential solutions in the face of these challenges, but its underlying neural mechanisms are not known. Using a combinatorial optimization paradigm, we show that rhesus macaques adopt distinct reasoning strategies and track the combinatorial upper bound--the best achievable outcome given the options examined so far. We recorded single units from the dorsolateral prefrontal cortex (DLPFC) - a structure central to higher cognition and complex behaviors - while the animals deliberated. Single DLPFC neurons encoded and updated the upper bound in real time, and the neuronal activity scaled with computational requirements. These findings uncover a neural correlate for combinatorial reasoning and reveal how the brain supports complex economic decisions.

neuroscience↗

bioRxiv2025

Rhesus macaque choices reveal that ambiguity aversion is driven by pessimistic probability estimates.

Economic uncertainty has multiple forms, including risk and ambiguity. Risk describes lotteries with well defined probabilities, whereas ambiguity describes cases where probabilities are not known. Given choices between risky and ambiguous alternatives, most decision makers prefer the known uncertainty of risk. This relationship, known as ambiguity aversion, holds even when both options have the same quantitative levels of uncertainty. Despite the prevalence of ambiguity in the natural world, the psychological and neural mechanisms that produce ambiguity aversion are not well understood. Here, we developed a nonhuman primate task to study ambiguity attitudes in the lab. We showed that the animals are strongly ambiguity averse, even when they could learn the true values of the ambiguous options through repeated trials. Then, to understand why they were ambiguity averse, we examined points of subjective equivalence between risky gambles and ambiguous alternatives. These experiments demonstrated that when the potential outcomes were large, the animals judged the probability of getting the better outcome far below the true probability of 0.5. Interestingly, we observed the opposite in small value gambles: the animals were overly optimistic about their chances of getting the better reward, even though the true probability was still 0.5. These inconsistent judgments about probabilities suggest that the animals do not hold consistent beliefs, and that the lack of consistent beliefs leads to suboptimal economic outcomes. These results provide a platform to study the neurophysiological bases of real-world decision making.

animal behavior and cognition↗

bioRxiv2025

A Multimodal Atlas Reveals the Anatomical Distribution of Medium Spiny Neuron Subtypes and a Novel RGS6+ Population in the Primate Striatum

The primate striatum and its principal neuron type, the medium spiny neuron (MSN), integrate cortical and subcortical signals related to movement, cognition, and emotion. These signals are processed through cell type specific circuits traditionally defined by MSN dopamine receptor expression. However, classification by dopamine receptor type alone fails to fully specify MSN diversity and falls short of capturing the functional complexity of the striatum. Here, we combined single-nucleus multi-omic sequencing and high-plex spatial transcriptomics to build a comprehensive atlas of MSNs in the macaque striatum. Using multi-omic sequencing, we profiled MSNs across four anatomically and functionally defined territories, and we mapped these subtypes back into their anatomical context by integrating the multi-omic data with [~]5.4 million spatially resolved cells sampled across the full rostral-caudal and dorsal-ventral extent of the striatum. This approach revealed two previously undocumented ventral striatum (VS) subtypes, D1-VS-RGS6 and D2-VS-RGS6, which are molecularly distinct from known ventral striatal MSNs yet share core limbic features. We also uncovered gradients in matrix-compartment cell types along the rostral-caudal axis. Finally, by integrating MSN subtype-specific transcriptomes and ATAC-seq-derived regulatory annotations with human GWAS data, we demonstrate strong, cell-type-specific enrichment of polygenic risk for Parkinsons disease, substance use disorders, and psychiatric and cognitive traits, including a striking association of D2-VS-RGS6 with schizophrenia and bipolar disorder. Together, this multimodal atlas provides a foundation for linking primate striatal cell types to circuit function and disease mechanisms. HIGHTLIGHTSO_LIMultimodal analysis of NHP striatum reveals heterogeneous cell type distribution C_LIO_LITwo previously uncharacterized MSN subtypes in the ventral striatum express RGS6 C_LIO_LIVentral striatum cell types exhibit similar characteristics across the Rostro-Caudal axis C_LIO_LINHP cell types show strong, cell type specific associations to genomic disease predictors C_LI

neuroscience↗

bioRxiv2025

Challenges in predicting chromatin accessibility differences between species

Enhancers are transcriptional regulatory elements that help drive phenotypic diversity, yet they often undergo rapid sequence evolution despite functional conservation, posing a challenge for predicting their function across species. Machine learning models that predict quantitative enhancer activity using DNA sequence have not previously been evaluated for their ability to predict quantitative differences across orthologous regions. Here, we trained convolutional neural networks (CNNs) on a regression task to predict chromatin accessibility, which is a proxy for enhancer activity, in the liver across five mammals, and we developed a novel framework to evaluate cross-species performance. We demonstrated that training on multiple species improves model generalization to both species used in training and held-out species. However, the models consistently achieved poor performance in predicting quantitative differences in accessibility between species at orthologous regions. Our study highlights the challenges in using regression models to predict chromatin accessibility changes between species.

genomics↗

bioRxiv2025

Combining Machine Learning and Multiplexed, In Situ Profiling to Engineer Cell Type and Behavioral Specificity

A promising strategy for the precise control of neural circuits is to use cis-regulatory enhancers to drive transgene expression in specific cells. However, enhancer discovery faces key challenges: low in vivo success rates, species-specific differences in activity, challenges with multiplexing adeno-associated viruses (AAVs), and the lack of spatial detail from single-cell sequencing. In order to accelerate enhancer discovery for the dorsal spinal cord--a region critical for pain and itch processing--we developed an end-to-end platform, ESCargoT (Engineered Specificity of Cargo Transcription), combining machine learning (ML)-guided enhancer prioritization, modular AAV assembly, and multiplexed, in situ screening. Using cross-species chromatin accessibility data, we trained ML models to predict enhancer activity in oligodendrocytes and in 15 dorsal horn neuronal subtypes. We first demonstrated that an initial enhancer, Excit-1, targeted excitatory dorsal horn neurons and drove reversal of mechanical allodynia in an inflammatory pain model. To enable parallel profiling of a 27-enhancer-AAV library delivered intraspinally in mice, we developed a Spatial Parallel Reporter Assay (SPRA) by integrating a novel Golden-Gate assembly pipeline with multiplexed, in situ screening. Regression adjustment for spatial confounding enabled specificity comparisons between enhancers, demonstrating the ability to screen enhancers targeting diverse cell types (oligodendrocytes, motoneurons, dorsal neuron subtypes) in one experiment. We then validated two candidates, targeting Exc-LMO3 and Exc-SKOR2 neurons, respectively. In a companion paper by Noh et al, our colleagues show that the functional specificity of the Exc-SKOR2-targeting enhancer, unlike Excit-1, is capable of blocking the sensation of chemical itch in mice. These enhancers were derived from the macaque genome but displayed functional sensitivity in mice. This platform enables spatially resolved, multiplexed in vivo enhancer profiling to accelerate discovery of cell-targeting tools and gene therapy development.

neuroscience↗

bioRxiv2025

Uncovering synaptic and cellular nanoarchitecture of brain tissue via seamless in situ trimming and milling for cryo-electron tomography

Cell-cell communication underlies all emergent properties of the brain, including cognition, learning and memory. The physical basis for these communications is the synapse, a multi-component structure requiring coordinated interactions between diverse cell types. However, many aspects of three-dimensional (3D) synaptic organization remain poorly understood. Here, we developed an approach, seamless in situ trimming and milling (SISTM), to reliably fabricate sufficiently thin lamellae for mapping of the 3D nanoarchitecture of synapses in mouse, monkey and human brain tissue under near-native conditions via cryo-electron tomography (cryo-ET). We validated SISTM in a mouse model of Huntingtons disease, demonstrating distinct 3D alterations to synaptic vesicles and mitochondria. By successfully applying SISTM to macaque brain, we described the 3D architecture of a tripartite synapse within the cortex. Subtomogram averaging (STA) enabled spatial mapping of astrocyte-neuron contacts within the tripartite synapse, revealing neurexin-neuroligin complexes as potential constituents that tether the two cell types. Finally, we showed that the defining features of synaptic nanoarchitecture were conserved across species and evident in human brain tissue obtained postmortem. Combining SISTM with cryo-ET and STA is a starting point for a new understanding of brain organization, disease-induced structural alterations and the development of rational, structure-guided therapeutics.

cell biology↗

bioRxiv2024

Cell Type Specific Enhancers for Dorsolateral Prefrontal Cortex.

The dorsolateral prefrontal cortex (DLPFC) is crucial to primate cognitive functions, but a paucity of cell type specific tools limits studies of DLPFC neurocomputational principles. Therefore, we set out to identify enhancers that fit inside Adeno-Associated Virus (AAV) vectors and that elicited functional, cell type specific gene expression in the non-human primate (NHP) DLPFC. We used single nucleus RNA-Seq and ATAC-Seq from rhesus macaque tissue samples to define DLPFC cell types and their associated open chromatin regions (OCRs). We trained machine learning (ML) models to recognize the unique regulatory grammar associated with each DLPFC neuron type, performed in silico screening of all OCRs, and identified candidate enhancers most likely to elicit cell type specific transgene expression in each neuron type. For layer 3 pyramidal neurons (L3PNs) and layer 5 extratelencephalic neurons (L5ETs), we cloned the top twelve identified candidates into AAVs and injected them into NHP DLPFC. In situ observation of enhancer-driven expression revealed the best performers, RMacL3-01 and RMacL5ET-01. We validated RMacL3-01 and RMacL5ET-01 using one-at-a-time injections in NHP DLPFC. RMacL3-01 restricted GFP expression to pyramidal neurons in layers 2 and 3, whereas RMacL5ET-01 restricted expression to POU3F1+ neurons in layer 5. RMacL3-01 elicited functional levels of channelrhodopsin expression that enabled optical activation of single- and multi-unit activity in NHP DLFPC. Together, these results and resources establish a solid foundation to study cell type specific principles of primate cognitive functions.

neuroscience↗

bioRxiv2024

Synaptic alterations in pyramidal cells following genetic manipulation of neuronal excitability in monkey prefrontal cortex

In schizophrenia, layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC) are thought to receive fewer excitatory synaptic inputs and to have lower expression levels of activity-dependent genes and of genes involved in mitochondrial energy production. In concert, these findings from previous studies suggest that DLPFC L3PNs are hypoactive in schizophrenia, disrupting the patterns of activity that are crucial for working memory, which is impaired in the illness. However, whether lower PN activity produces alterations in inhibitory and/or excitatory synaptic strength has not been tested in the primate DLPFC. Here, we decreased PN excitability in rhesus monkey DLPFC in vivo using adeno-associated viral vectors (AAVs) to produce Cre recombinase-mediated overexpression of Kir2.1 channels, a genetic silencing tool that efficiently decreases neuronal excitability. In acute slices prepared from DLPFC 7-12 weeks post-AAV microinjections, Kir2.1-overexpressing PNs had a significantly reduced excitability largely attributable to highly specific effects of the AAV-encoded Kir2.1 channels. Moreover, recordings of synaptic currents showed that Kir2.1-overexpressing DLPFC PNs had reduced strength of excitatory synapses whereas inhibitory synaptic inputs were not affected. The decrease in excitatory synaptic strength was not associated with changes in dendritic spine number, suggesting that excitatory synapse quantity was unaltered in Kir2.1-overexpressing DLPFC PNs. These findings suggest that, in schizophrenia, the excitatory synapses on hypoactive L3PNs are weaker and thus might represent a substrate for novel therapeutic interventions. Significance StatementIn schizophrenia, dorsolateral prefrontal cortex (DLPFC) pyramidal neurons (PNs) have both transcriptional and structural alterations that suggest they are hypoactive. PN hypoactivity is thought to produce synaptic alterations in schizophrenia, however the effects of lower neuronal activity on synaptic function in primate DLPFC have not been examined. Here, we used, for the first time in primate neocortex, adeno-associated viral vectors (AAVs) to reduce PN excitability with Kir2.1 channel overexpression and tested if this manipulation altered the strength of synaptic inputs onto the Kir2.1-overexpressing PNs. Recordings in DLPFC slices showed that Kir2.1 overexpression depressed excitatory (but not inhibitory), synaptic currents, suggesting that, in schizophrenia, the hypoactivity of PNs might be exacerbated by reduced strength of the excitatory synapses they receive.

neuroscience↗

bioRxiv2024

An open-source MRI compatible frame for multimodal presurgical mapping in macaque and capuchin monkeys

HighlightsO_LIWe present a compact MRI-compatible stereotaxic frame for large nonhuman primates. C_LIO_LIThe design is 3D printable, inexpensive, and matches size of an adult human head. C_LIO_LIEnabled real-time, accurate, MRI-guided deep-brain viral vector injection. C_LIO_LIFacilitated multimodal alignment for deep-brain electrophysiology planning. C_LIO_LIAll computer-aided-design files are modularized and publicly available and editable. C_LI BackgroundHigh-precision neurosurgical targeting in nonhuman primates (NHPs) often requires presurgical anatomy mapping with noninvasive neuroimaging techniques (MRI, CT, PET), allowing for translation of individual anatomical coordinates to surgical stereotaxic apparatus. Given the varied tissue contrasts that these imaging techniques produce, precise alignment of imaging-based coordinates to surgical apparatus can be cumbersome. MRI-compatible stereotaxis with radiopaque fiducial markers offer a straight-forward and reliable solution, but existing commercial options do not fit in conformal head coils that maximize imaging quality. New methodWe developed a compact MRI-compatible stereotaxis suitable for a variety of NHP species (Macaca mulatta, Macaca fascicularis, and Cebus apella) that allows multimodal alignment through technique-specific fiducial markers. Comparison with existing methodsWith the express purpose of compatibility with clinically available MRI, CT, and PET systems, the frame is no larger than a human head, while allowing for imaging NHPs in the supinated position. This design requires no marker implantation, special software, or additional knowledge other than the operation of a common large animal stereotaxis. ResultsWe demonstrated the applicability of this 3D-printable apparatus across a diverse set of experiments requiring presurgical planning: 1) We demonstrate the accuracy of the fiducial system through a within-MRI cannula insertion and subcortical injection of viral vectors. 2) We also demonstrated accuracy of multimodal (MRI and CT) alignment and coordinate transfer to guide a surgical robot electrode implantation for deep-brain electrophysiology. ConclusionsThe computer-aided design files and engineering drawings are publicly available, with the modular design allowing for low cost and manageable manufacturing.

bioengineering↗

bioRxiv2020

The Regulatory Evolution of the Primate Fine-Motor System

In mammals, fine motor control is essential for skilled behavior, and is subserved by specialized subdivisions of the primary motor cortex (M1) and other components of the brains motor circuitry. We profiled the epigenomic state of several components of the Rhesus macaque motor system, including subdivisions of M1 corresponding to hand and orofacial control. We compared this to open chromatin data from M1 in rat, mouse, and human. We found broad similarities as well as unique specializations in open chromatin regions (OCRs) between M1 subdivisions and other brain regions, as well as species- and lineage-specific differences reflecting their evolutionary histories. By distinguishing shared mammalian M1 OCRs from primate- and human-specific specializations, we highlight gene regulatory programs that could subserve the evolution of skilled motor behaviors such as speech and tool use.

neuroscience↗

bioRxiv2020

Transcriptional Diversity of Medium Spiny Neurons in the Primate Striatum

The striatum is the interface between dopamine reward signals and cortico-basal ganglia circuits that mediate diverse behavioral functions. Medium spiny neurons (MSNs) constitute the vast majority of striatal neurons and are traditionally classified as direct- or indirect-pathway neurons. However, that traditional model does not explain the anatomical and functional diversity of MSNs. Here, we defined molecularly distinct MSN types in the primate striatum, including (1) dorsal striatum MSN types associated with striosome and matrix compartments, (2) ventral striatum types associated with the nucleus accumbens shell and olfactory tubercle, and (3) an MSN-like type restricted to -opioid receptor rich islands in the ventral striatum. These results lay the foundation for achieving cell type-specific transgenesis in the primate striatum and provide a blueprint for investigating circuit-specific processing.

neuroscience↗

bioRxiv2020

scAAVengr: Single-cell transcriptome-based quantification of engineered AAVs in non-human primate retina

Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to quantify efficiency of AAV-mediated gene expression across all cell types. scAAVengr allows for definitive, head-to-head comparison of vectors in the same animal. To demonstrate proof-of-concept for the scAAVengr workflow, we quantified - with cell-type resolution - the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. These results validate scAAVengr as a powerful method for development of AAV vectors.

bioengineering↗

bioRxiv2019

Rare Rewards Amplify Dopamine Learning Responses

Dopamine neurons drive learning by coding reward prediction errors (RPEs), which are formalized as subtractions of predicted values from reward values. Subtractions accommodate point estimate predictions of value, such as the average value. However, point estimate predictions fail to capture many features of choice and learning behaviors. For instance, reaction times and learning rates consistently reflect higher moments of probability distributions. Here, we demonstrate that dopamine RPE responses code probability distributions. We presented monkeys with rewards that were drawn from the tails of normal and uniform reward size distributions to generate rare and common RPEs, respectively. Behavioral choices and pupil diameter measurements indicated that monkeys learned faster and registered greater arousal from rare RPEs, compared to common RPEs of identical magnitudes. Dopamine neuron recordings indicated that rare rewards amplified RPE responses. These results demonstrate that dopamine responses reflect probability distributions and suggest a neural mechanism for the amplified learning and enhanced arousal associated with rare events.

neuroscience↗