Biology subjects

Peterka, D. S.

Publications and source records attributed to Peterka, D. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Photon-Resolved Excitation-Denoised (PRED) Three-Photon Imaging Improves Detection of Neuronal Activity in Awake and Behaving Mice

Three-photon microscopy (3PM) has enabled the optical access of neurons [~]500-1500 {micro}m below the brain surface but has been limited to slow imaging frame rates or small total imaged areas by competing constraints: a high peak power requirement for nonlinear excitation, and the need to limit total average power. Additionally, the high sensitivity to laser power fluctuations and the inherently dim signals introduce additional challenges and add error. When combined with other effects, like brain motion in behaving animals, 3P imaging of neuronal activity during animal behavior has remained inadequate. Herein, we systematically address these limitations by 1) carefully balancing scanning speed with power requirements, 2) using a deeply cooled silicon photomultiplier detector and Bayesian statistics-based processing (PRED-correction) to reduce excess noise, and 3) by spatiotemporal shaping of excitation pulses. Our improvements enable rapid (20-30Hz) imaging of calcium activity in the dorsal hippocampal dentate gyrus of behaving mice, allowing the identification of spatially tuned neurons and the recapitulation of established functional properties across different cell types in this brain region. PRED-3P imaging provides a new approach to functional characterization of cells deep in the brain that were previously inaccessible to two-photon imaging.

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bioRxiv2026

The External Globus Pallidus is a Basal Ganglia Output Hub with Action-Specific Circuits

The external globus pallidus (GPe) is traditionally viewed as a homogeneous relay in the indirect basal ganglia pathway that broadly suppresses movement. Using whole-brain anterograde axon mapping, rabies tracing, single-neuron reconstruction, and single-nucleus RNA sequencing, we reveal that the GPe is a major basal ganglia output nucleus, composed of anatomically and molecularly distinct populations. Besides the neurons with canonical projections, and projections to striatum and cortex, the GPe contains specific neuronal populations that target the thalamus and brainstem directly, including the parafascicular thalamus (GPePf) and the pedunculopontine nucleus (GPePPN). These projection-defined subpopulations exhibit distinct behavioral functions. GPe-PPN neurons are selectively suppressed at locomotor onset, and bidirectional manipulation of this pathway is sufficient to promote or suppress locomotion. Notably, D2-MSN stimulation upstream of these neurons evokes locomotion, and co-activation of GPePPN pathway blocks this D2-MSN-evoked locomotion, demonstrating a disinhibitory circuit for action stemming from D2-MSNs. In contrast, GPePf neurons are not engaged during locomotion but are selectively recruited during skilled forelimb actions, and their activation disrupts forelimb movements without affecting locomotion. Together, these findings establish the GPe as a basal ganglia output hub composed of modules that mediate distinct behaviors. This organization revises canonical models of the indirect pathway by demonstrating that D2-MSNs can also facilitate, rather than only suppress, movement depending on the downstream GPe output channels they engage. In briefThe external globus pallidus (GPe), classically considered a relatively homogeneous relay within the indirect basal ganglia pathway, is revealed here as a major basal ganglia output hub. Distinct GPe subpopulations, defined by transcriptomic identity and projection target, form parallel channels to cortical, thalamic and brainstem structures with action-specific functions. These findings uncover a disinhibitory mechanism through which D2-MSNs can facilitate behavior, revising canonical models of basal ganglia function. HighlightsBrain-wide mapping reveals the GPe as a major basal ganglia output node GPe contains novel cell types defined by transcriptomic identity and projection specificity Projection-specific GPe circuits differentially control locomotion and skilled forelimb actions D2-MSNs can disinhibit downstream targets via specific GPe projections and promote action.

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bioRxiv2026

Global context rapidly shapes sensory responses in V1

Context modulates sensory processing in the cerebral cortex by suppressing responses to expected stimuli and enhancing responses to unexpected ones. Recent proposals argue that early sensory areas such as primary visual cortex (V1) are shaped only by local context, including recent stimulus history, whereas modulation by global context, such as learned temporal structure, is present exclusively in higher cortical areas. This view is incompatible with predictive coding theories. To directly dissociate local and global contextual influences, we used a global/local oddball paradigm in which mice viewed five-item sequences. Across conditions, sequence structure was held constant while stimulus identity and predictability were selectively manipulated, allowing the isolation of response modulations due to local deviance, global expectation, and stimulus repetition independently. In the canonical sequence (AAAA-B), B is locally deviant but globally predictable. Using two-photon calcium imaging and LFP recordings in mouse V1, we found that global predictability abolished context modulation: responses to B were equivalent to those evoked by a random sequence control (e.g., CDEAB). This effect emerged rapidly, after only <10 sequence repetitions, demonstrating fast learning of global structure. When the stimulus was globally deviant, either by replacing B with a novel stimulus (AAAA-C) or by presenting B unpredictably in a standard oddball paradigm, V1 exhibited robust response enhancement. These effects required feedback from anterior cingulate area (ACa), establishing a causal role for higher cortical circuits in conveying global predictions to V1. Strikingly, when an additional A replaced B (AAAA-A), responses were strongly suppressed despite global deviance, indicating that stimulus-specific adaptation may constrain the expression of global prediction error signals in early sensory cortex. HighlightsGlobal context rapidly modulates neural responses in V1 Global deviations, but not predictable local deviants, elicit enhanced responses Downstream brain region ACa is necessary for global context modulation in V1 Standard oddball paradigms involve higher-order contextual modulation

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bioRxiv2025

Striatal ensembles specify and control granular forelimb actions

The striatum is classically thought to modulate action vigor and reinforcement, but whether it specifies and controls ongoing actions at a fine scale remains unclear. Here, we test whether striatal activity can resolve and control actions as granular as distinct patterns of muscle co-contraction within the same limb. We developed a closed-loop system combining two-photon imaging and holographic optogenetics to identify and manipulate action-specific striatal ensembles in real time. Mice performed isometric push or pull actions engaging the same forelimb muscles with different activation patterns. Ensembles of both D1- and D2-spiny projection neurons encoded action identity with equal fidelity. Causal stimulation of small ensembles biased ongoing force, but only when the ensembles encoded the ongoing action. These results show that the striatum specifies and controls ongoing actions in real time at a previously unappreciated granularity, redefining its role in motor control.

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bioRxiv2025

ALM enables contextual decision-making via dynamic reconfiguration of local circuits

Cognitive operations often require flexible implementation of stimulus-response contingencies, depending on context. We developed an olfactory task in which mice learned to associate a test odor with a directional lick response, conditional on a preceding context odor drawn from a different odor set. Two-photon imaging revealed that anterior lateral motor cortex (ALM) contains distinct populations encoding context, test odors, and choice. Optogenetic silencing during the context and delay periods impaired performance, suggesting that ALM contributes to configuring the appropriate contingency. Although context odors that instructed the same mapping were represented by separate populations, their influence converged at the level of choice-selective neurons. A subpopulation of these neurons exhibited dual selectivity for context and choice, forming what we term "contingency neurons." These findings suggest that ALM supports flexible behavior not by abstracting over context cues, but by dynamically reconfiguring local circuits to route sensory input to the appropriate motor output.

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bioRxiv2025

Fast photostimulus optimization for holographic control of neural ensemble activity in vivo

Determining the intricate structure and function of neural circuits requires the ability to precisely manipulate circuit activity. Two-photon holographic optogenetics has emerged as a powerful tool for achieving this via flexible excitation of user-defined neural ensembles. However, the precision of two-photon optogenetics has been constrained by off-target stimulation, an effect where proximal non-target neurons can be unintentionally activated due to imperfect spatial confinement of light onto target neurons. Here, we introduce a real-time computational method to mitigating off-target stimulation that first empirically samples each neurons sensitivity to stimulation at proximal locations, and then optimizes stimulation sites using a fast, interpretable model based on adaptive non-negative basis function regression (NBFR). NBFR is highly scalable, completing model fitting for hundreds of neurons in just a few seconds and then optimizing stimulation sites in several hundred milliseconds per stimulus - fast enough for most closed-loop behavioral experiments. We characterize the performance of our approach in both simulations and in vivo experiments in mouse hippocampus, showing its efficacy under realistic experimental conditions. Our results thus establish NBFR-based photostimulus optimization as an important addition to an emerging computational toolkit for precise yet scalable holographic optogenetics.

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bioRxiv2025

Dopamine release effects on striatal blood oxygenation and whole brain plasticity underlying associative learning

Dopaminergic signaling in the nucleus accumbens (NAc) is central to reward-based learning, but its relationship to brain-wide hemodynamics remains unclear. Using concurrent fMRI and dopamine photometry in awake, behaving mice, we reveal that associative learning induces a gradual temporal shift in NAc blood oxygenation responses that mirrors dopamine release dynamics. This shift emerges with cue-reward learning and extends across a distributed network including prefrontal, insular, and hypothalamic regions. Further, dopamine transients tightly correspond with local BOLD signals, and variations in reward value modulate delayed BOLD responses in both the NAc and additional subcortical structures. Removing dopaminergic contributions abolishes this reward-related modulation, demonstrating that BOLD signals encode dopaminergic value prediction. These findings establish a mechanistic link between dopamine signaling and widespread neural plasticity during learning.

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bioRxiv2025

Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons

The basal ganglia and midbrain dopaminergic systems are critical for motor control, reward processing, and reinforcement learning, with dysfunction in these systems implicated in numerous neurodegenerative and neuropsychiatric disorders. To enable precise genetic targeting of key neuronal populations, we generated and characterized five knock-in mouse lines: Drd1-Cre, Adora2a-Cre, Drd1-FlpO, Adora2a-FlpO, and DAT-FlpO. These lines allow for Cre-or FlpO-mediated recombination in dopamine D1 receptor-expressing spiny projection neurons (SPNs), adenosine A2a receptor-expressing SPNs, and dopamine transporter (DAT)-expressing neurons in the midbrain. Histological analyses confirmed recombinase activity in expected brain regions, and whole-cell electrophysiological recordings validated the intrinsic excitability profiles of each neuronal subpopulation. These tools provide high specificity and reliability for studying basal ganglia circuitry and dopaminergic neurons. By enabling targeted manipulations, these openly available knock-in lines will advance research into the neural mechanisms underlying motor control, reward, and neuropsychiatric diseases.

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bioRxiv2024

Recurrent Connectivity Shapes Spatial Coding in Hippocampal CA3 Subregions

Stable and flexible neural representations of space in the hippocampus are crucial for navigating complex environments. However, how these distinct representations emerge from the underlying local circuit architecture remains unknown. Using two-photon imaging of CA3 subareas during active behavior, we reveal opposing coding strategies within specific CA3 subregions, with proximal neurons demonstrating stable and generalized representations and distal neurons showing dynamic and context-specific activity. To test the causal role of excitatory connectivity in neural computation, we employed a genetic manipulation approach in which local disruption of glutamatergic synaptic transmission impaired context-specific spatial coding in distal CA3. Aligned with these experimental results, we show in artificial neural network models that varying the recurrence level causes these differences in coding properties to emerge. We confirmed the contribution of recurrent connectivity to functional heterogeneity by characterizing the representational geometry of neural recordings and comparing it with theoretical predictions of neural manifold dimensionality. Our results indicate that local circuit organization, particularly recurrent connectivity among excitatory neurons, plays a key role in shaping complementary spatial representations within the hippocampus. HighlightsO_LIProximodistal CA3 subregions implement complementary coding strategies in relation to time and context C_LIO_LIDisrupting excitatory recurrence in distal CA3 impairs context-discriminative neural coding C_LIO_LISparse and dense neural networks capture the functional heterogeneity of CA3 subcircuits C_LIO_LIRecurrence level tunes the geometry of neural manifolds both in vivo and in silico C_LI

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bioRxiv2024

A lasting impact of serotonergic psychedelics on visual processing and behavior

Serotonergic psychedelics (e.g., psilocybin, LSD) have potential to treat psychiatric disorders, with therapeutic effects lasting days to weeks after a single dose. Prominent theories suggest that psychedelics have a lasting effect on hierarchical brain circuits, reducing top-down influence on information processing to facilitate an unbiased, bottom-up reassessment of the world, but direct and concrete evidence for such an effect is lacking. Here we directly tested this hypothesis in both humans and mice, assessing predictive processing in the fronto-visual system in the days after a single psychedelic exposure. Individuals who recently (<3 weeks) used 5-HT2AReceptor agonist psychedelics (psilocybin, LSD) were assessed via electroencephalography (EEG) and electrooculography recordings during a saccadic prediction task and compared to age- and sex-matched non-users. Compared to non-users, recent psychedelic users produced fewer fast saccades and less suppression of EEG delta/theta power to predictively presented stimuli, pointing to a disruption of predictive processing. These changes correlated with time since psychedelic use and were replicated in a second cohort taking a different serotonergic psychedelic (5-MeO-DMT). Direct recordings of primary visual cortex (V1) in mice administered psilocybin (1 mg/kg) evinced a similar loss of predictive suppression 24-hrs after the dose. This coincided with weakened top-down modulation of V1 from anterior cingulate area (ACa), a subregion of medial prefrontal cortex, along with clear spine growth in ACa neurons that project to V1. These results suggest that psychedelic-induced neural plasticity serves to reorganize feedback circuits in the cortex and relax top-down influence on bottom-up sensory processing - an effect that persists beyond the acute exposure period and may underlie a therapeutic window.

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bioRxiv2024

Standardised Measurements for Monitoring and Comparing Multiphoton Microscope Systems

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

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bioRxiv2024

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via RiboTag, morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

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bioRxiv2023

maskNMF: A denoise-sparsen-detect approach for extracting neural signals from dense imaging data

A number of calcium imaging methods have been developed to monitor the activity of large populations of neurons. One particularly promising approach, Bessel imaging, captures neural activity from a volume by projecting within the imaged volume onto a single imaging plane, therefore effectively mixing signals and increasing the number of neurons imaged per pixel. These signals must then be computationally demixed to recover the desired neural activity. Unfortunately, currently-available demixing methods can perform poorly in the regime of high imaging density (i.e., many neurons per pixel). In this work we introduce a new pipeline (maskNMF) for demixing dense calcium imaging data. The main idea is to first denoise and temporally sparsen the observed video; this enhances signal strength and reduces spatial overlap significantly. Next we detect neurons in the sparsened video using a neural network trained on a library of neural shapes. These shapes are derived from segmented electron microscopy images input into a Bessel imaging model; therefore no manual selection of "good" neural shapes from the functional data is required here. After cells are detected, we use a constrained non-negative matrix factorization approach to demix the activity, using the detected cells shapes to initialize the factorization. We test the resulting pipeline on both simulated and real datasets and find that it is able to achieve accurate demixing on denser data than was previously feasible, therefore enabling faithful imaging of larger neural populations. The method also provides good results on more "standard" two-photon imaging data. Finally, because much of the pipeline operates on a significantly compressed version of the raw data and is highly parallelizable, the algorithm is fast, processing large datasets faster than real time.

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bioRxiv2023

A frontosensory circuit for visual context processing is synchronous in the theta/alpha band

Visual processing is strongly influenced by context. Stimuli that deviate from contextual regularities elicit augmented responses in primary visual cortex (V1). These heightened responses, known as "deviance detection," require both inhibition local to V1 and top-down modulation from higher areas of cortex. Here we investigated the spatiotemporal mechanisms by which these circuit elements interact to support deviance detection. Local field potential recordings in mice in anterior cingulate area (ACa) and V1 during a visual oddball paradigm showed that interregional synchrony peaks in the theta/alpha band (6-12 Hz). Two-photon imaging in V1 revealed that mainly pyramidal neurons exhibited deviance detection, while vasointestinal peptide-positive interneurons (VIPs) increased activity and somatostatin-positive interneurons (SSTs) decreased activity (adapted) to redundant stimuli (prior to deviants). Optogenetic drive of ACa-V1 inputs at 6-12 Hz activated V1-VIPs but inhibited V1-SSTs, mirroring the dynamics present during the oddball paradigm. Chemogenetic inhibition of VIP interneurons disrupted ACa-V1 synchrony and deviance detection responses in V1. These results outline spatiotemporal and interneuron-specific mechanisms of top-down modulation that support visual context processing.

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bioRxiv2019

Prolonged anesthesia alters brain synaptic architecture

Prolonged medically-induced coma (pMIC), a procedure performed in millions of patients worldwide, leads to cognitive impairment, yet the underlying brain mechanism remains unknown. No experimental studies of medically-induced coma (MIC) exceeding ~6 hours exist. For MIC of less than 6 hours, studies in developing rodents have documented transient changes of cortical synapse formation. However, in adulthood, cortical synapses are thought to become stabilized. Here, we establish pMIC (up to 24 hrs) in adolescent and mature mice, and combine repeated behavioral object recognition assessments with longitudinal two-photon imaging of cortical synapses. We find that pMIC affects cognitive function, and is associated with enhanced synaptic turnover, generated by enhanced synapse formation during pMIC, while the post-anesthetic period is dominated by synaptic loss. These results carry profound implications for intensive medical care, as they point out at significant structural side effects of pMIC on cortical brain synaptic architecture across age levels.

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bioRxiv2019

An amygdala circuit mediates experience-dependent momentary exploratory arrests

Exploration of novel environments ensures survival and evolutionary fitness. This behavior is expressed through exploratory bouts and arrests, which change dynamically based on experience. Neural circuits mediating exploratory behavior should therefore integrate experience and use it to select the proper behavioral output. Using a spatial exploration assay, we uncovered an experience-dependent increase of momentary arrests in visited locations where animals previously arrested. Quantitative analyses of neuronal calcium activity in freely-exploring mice revealed that a large neuronal ensemble in basolateral amygdala is active during self-paced behavioral arrests. This ensemble was recruited in an experience-dependent manner, and closed-loop optogenetic manipulation of these neurons revealed that they are sufficient and necessary to drive experience-dependent arrests. Additionally, we found that neurons in the basolateral amygdala projecting to central amygdala mediate these momentary arrests. These findings uncover an amygdala circuit that mediates momentary exploratory arrests in familiar places, without changing place preference or anxiety/fear-like behaviors.

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bioRxiv2018

Seizures start as silent microseizures by neuronal ensembles

Understanding seizure formation and spread remains a critical goal of epilepsy research. While many studies have documented seizure spread, it remains mysterious how they start. We used fast in-vivo two-photon calcium imaging to reconstruct, at cellular resolution, the dynamics of focal cortical seizures as they emerge in epileptic foci (intrafocal), and subsequently propagate (extrafocal). We find that seizures start as intrafocal coactivation of small numbers of neurons (ensembles), which are electrographically silent. These silent \"microseizures\" expand saltatorily until they break into neighboring cortex, where they progress smoothly and first become detectable by LFP. Surprisingly, we find spatially heterogeneous calcium dynamics of local PV interneuron sub-populations, which rules out a simple role of inhibitory neurons during seizures. We propose a two-step model for the circuit mechanisms of focal seizures, where neuronal ensembles first generate a silent microseizure, followed by widespread neural activation in a travelling wave, which is then detected electrophysiologically.

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bioRxiv2017

Three-Dimensional Two-Photon Optogenetics And Imaging Of Neural Circuits In Vivo

We demonstrate a holographic system for simultaneous three-dimensional (3D) two-photon stimulation and imaging of neural activity in the mouse neocortex in vivo with cellular resolution. Dual two-photon excitation paths are implemented with independent 3D targeting for calcium imaging and precision optogenetics. We validate the usefulness of the microscope by photoactivating local pools of interneurons in awake mice visual cortex in 3D, which suppress the nearby pyramidal neurons response to visual stimuli.

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