Neurophotonics and Imaging Transcriptomics

May 15-22, 2027

Director: Mark Schnitzer

Stanford University, Howard Hughes Medical Institute, Stanford, USA

Faculty:

Karel Svoboda, Allen Institute for Neural Dynamics, Seattle, USA

Michael Lin, Weill Cornell Medicine, New York, USA

Hillel Adesnik, University of California, Berkeley, USA

Valentina Emiliani, Vision Institute, INSERM, Paris, France

Hongkui Zeng, Allen Institute for Brain Science, Seattle, USA

Misha Ahrens, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, USA

Mark Schnitzer, Stanford University, Howard Hughes Medical Institute, Stanford, USA

The last decade has witnessed a veritable explosion of optical methods for monitoring and manipulating brain activity. New fluorescent indicators, optogenetic actuators of neural dynamics, and microscopes for imaging neural activity patterns at cellular resolution and at large scales have collectively provided neuroscientists with game-changing capabilities for visualizing and experimentally perturbing brain dynamics in awake behaving animals.

This Advanced Course explores these cutting-edge topics in neurophotonics and features tutorial lectures on both foundational principles and the most recent advances in the use of light to study brain dynamics, across scales from single neurons to ensembles of ~100,000 cells or more. The panel of world experts will cover multiple topics, including fluorescent indicators of neural calcium, voltage, neurotransmission and neuromodulatory dynamics; optogenetic excitation and inhibition of neural activity; imaging of electrical oscillations and brain waves; tabletop and head-mounted miniature microscopes that use one-, two- or three-photon fluorescence excitation for imaging neural dynamics in head-fixed or freely behaving animals; adaptive optical, multi-plane, high-speed, multi-beam and volumetric imaging of large-scale neural activity patterns; and holographic and two-photon optogenetic methods for precisely manipulating neural firing.

Overall, the Advanced Course will allow students and scholars presently using or planning to use optical techniques to gain a deeper understanding of the conceptual underpinnings and recent advancements in the rapidly emerging, exciting field of neurophotonics.

Valentina Emiliani

Precise neuronal circuit interrogation using two-photon holographic illumination, optogenetics and voltage imaging

Optogenetic neuronal targeting combined with single-photon wide-field illumination has already demonstrated immense potential in neuroscience, enabling optical control of entire neuronal networks and elucidating their roles in specific behaviors. However, to understand how individual neurons or subsets of neurons control specific behaviors, how functionally identical neurons connect during particular tasks, or how neuronal circuits dynamically modify behaviors in real time, more advanced approaches requires more sophisticated approaches enabling the activation of neuronal circuits with single-cell resolution and millisecond temporal accuracy. Over the past few years, this need has driven the development of flexible optical methods for two-photon (2P) optogenetic activation, which enable the interrogation of brain circuits in depth with high spatial and temporal resolution over large volumes. We will review the most commonly used methods for 2P optogenetics, including laser spiral scanning, holographic light multiplexing, and temporal focusing. We will then present the most recent variants of these systems, enabling the scaling up of achievable targets through fast sequential light targeting, or enabling circuit manipulation in freely moving mice through 2P-holographic endoscopy. Finally, we will demonstrate how the same holographic system used for 2P optogenetics also enables 2P scanless voltage recording from multiple targets in the in vivo mouse brain.

Hillel Adesnik

Optogenetics and illumination schemes: next-generation opsins for neural control

The lectures will focus on advanced topics in microbial optogenetics and illumination schemes for exploiting ultrapotent next-generation opsins for massive-scale, cellular-resolution neural control. First, I will provide a general background on optogenetics, microbial opsins, and their key features that make different variants optimal for specific applications. Then I will discuss visible-light and two-photon excitation schemes that enable fast, volumetric, cellular-scale optogenetics in the brains of intact animals. Finally, I will share our laboratory’s latest work on bidirectional optogenetics, all-optical electrophysiology, and new systems for mesoscale optical control of multiple brain areas simultaneously.

Hongkui Zeng

Diversity and dynamics of mammalian brain cell types

To understand brain function and how its dysfunction leads to disease, it is essential to uncover its cell-type composition, how cell types connect, and their roles in circuit function. At the Allen Institute, we generated a comprehensive, high-resolution transcriptomic and spatial cell type atlas of the whole adult mouse brain, comprising >5,300 hierarchically organised clusters. To reveal evolutionary conservation and divergence of cell types in the mammalian brain, we are creating similarly high-resolution cell type atlases for human and non-human primate brains and computationally integrating them into cross-species aligned cell type taxonomies.

Building on these foundational reference atlases, we have investigated dynamic changes in the transcriptomic profiles of specific cell types in the developing and aging brain, as well as under various behavioral and drug-treated conditions. In the aging mouse brain, using brain-wide single-cell transcriptomic profiling, we uncovered cell-type-specific transcriptomic signatures of reduced neuronal structure and function, alongside increased immune response and inflammation. We further identified a potential hotspot for aging involving specific hypothalamic cell types that regulate energy homeostasis and exhibit both decreased neuronal function and increased immune response, suggesting a connection among metabolism, neuroinflammation, and aging. As a first deep characterization of brain development, we generated a transcriptomic and epigenomic cell-type atlas of the developing mouse visual cortex, with dense temporal sampling from E11.5 to P28. We reconstructed a transcriptomic developmental trajectory map of all excitatory, inhibitory, and non-neuronal cell types in the visual cortex, revealing continuous cell-type diversification throughout the pre- and post-natal stages of cortical development. We have now extended the developmental study to the whole mouse brain and begun to reveal age-dependent transcriptomic, epigenomic, and spatial dynamics across brain-wide neuronal and non-neuronal cell types.

By measuring cell type-specific expression of immediate early genes (IEGs) and other differentially expressed genes (DEGs), we identified highly specific transcriptomic cell types (T-types) activated by innate behaviors across the hypothalamus and extended amygdala (HY-EA). We also identified sex-dominant cell types enriched in HY-EA, validated by two whole-forebrain MERFISH datasets in each sex, and shown by IEG analysis to be preferentially activated during sexually dimorphic behaviors. In a brain-wide survey of T-types responsive to acute or chronic administration of addictive or psychoactive compounds, we uncovered distinct patterns of cell-type activation and differential gene expression across many brain regions in response to different drugs. These findings will facilitate deeper investigation of the neural circuits governing diverse behavioral phenotypes and offer new insights into the mechanisms underlying cell-state dynamics in brain function and disease.

Karel Svoboda

Cell type-specific neurophysiology

Every biological organ comprises diverse cell types that work together to keep the body running. The brain is no exception. Brain disorders can be traced to a single cell type, such as dopamine neurons in Parkinson’s. Over the last decade, large-scale projects based on single-cell RNA sequencing have provided taxonomies of the brain’s cell types in mice and primates, including humans.

A cell type’s importance lies in what it does in a living brain. Electrophysiology is unmatched on two axes that matter for watching neurons in action: millisecond timing and access to thousands of neurons anywhere in the brain. Cellular imaging excels at measuring neural activity densely within the imaged area, tracking activity over long time scales, and detecting rare cell types. We have a more or less complete list of cell types, and advanced methods to listen in on their activity. I will describe advances from the Allen Institute on how to align neurophysiology with cell types.

Cell type-specific electrophysiology and cellular imaging require different approaches. For electrophysiology, we use optotagging. This approach involves expressing light-activated proteins in genetically defined cell types; brief pulses of light during recording reveal which neurons belong to which type. Optotagging has recently been turbocharged by two technologies: Neuropixels Opto, which combines high-density recording with random-access delivery of dual color light to small volumes of tissue in the vicinity of recorded neurons; and enhancer AAVs, which enable efficient delivery of optogenetic reagents to genetically defined cell types. Optotagging provides limited multiplexing (a handful of cell types in a brain). However, cell type-specific recordings from optotagging can be used as training data to develop ML classifiers based on cell type-specific spike shapes and firing patterns – a cell type look-up table (ctLUT). The ctLUT allows classification of recorded neurons without optotagging and potentially enables deep multiplexing for extracellular neurophysiology.

For imaging, we employ retrospective multiplexed single-molecule fluorescent in situ hybridization (mFISH) to identify the genetic identity of nearly all imaged neurons (thousands per experiment). We have developed pipelines based on mFISH in 3D-expanded tissue and on commercial spatial transcriptomics platforms. Importantly, these methods allow classification of cell types at the finest resolution of the cell-type taxonomy. I will discuss the state of the art of cell-type-specific electrophysiology and imaging and highlight current limitations and challenges.

Misha Ahrens

Whole-brain and whole-body computation for behavior and physiology

Flexible behavior arises from brain and brain-body circuits whose collective dynamics depend on context, physiological state, and precise wiring. Understanding these systems benefits from measuring them as a whole. To this end, as part of a collaboration, we are assembling a dense connectome of a larval zebrafish brain, together with light-sheet whole-brain activity imaging and behavior in the same animal. This allows brain-wide dynamics to be interpreted in terms of the synaptic circuits that generate them. Also, we asked how animals manage metabolic resources during behavior. Swimming drives internal hypoxia with a multi-second delay, yet behavioral adaptations occur before hypoxia appears, indicating predictive rather than reactive control. Noradrenergic neurons in the medulla oblongata sum brain oxygen levels with efference copies of swim actions; astroglia integrate this input into a prolonged calcium elevation that tracks the oxygen cost of recent actions and rises ten seconds before oxygen falls, reorganizing brain-wide activity to suppress locomotion and promote respiration to preempt, as in allostasis, physiological stress. Finally, extending beyond the brain, we developed whole-body cellular imaging to record second-timescale activity of cells throughout the entire vertebrate body at cellular resolution. It revealed chondrocyte responses to cold, rhythmic traveling waves along the renal nephron, previously unknown muscle synergies and muscle-organ interactions, and brainstem-controlled redistribution of body-wide blood flow. Combined with optogenetics, it enables all-optical causal dissection of brain-body interactions.