Cell-type-specific population dynamics of diverse reward computations.
2022; 185 (19): 3568
Computational analysis of cellular activity has developed largely independently of modern transcriptomic cell typology, but integrating these approaches may be essential for full insight into cellular-level mechanisms underlying brain function and dysfunction. Applying this approach to the habenula (a structure with diverse, intermingled molecular, anatomical, and computational features), we identified encoding of reward-predictive cues and reward outcomes in distinct genetically defined neural populations, including TH+ cells and Tac1+ cells. Data from genetically targeted recordings were used to train an optimized nonlinear dynamical systems model and revealed activity dynamics consistent with a line attractor. High-density, cell-type-specific electrophysiological recordings and optogenetic perturbation provided supporting evidence for this model. Reverse-engineering predicted how Tac1+ cells might integrate reward history, which was complemented by invivo experimentation. This integrated approach describes a process by which data-driven computational models of population activity can generate and frame actionable hypotheses for cell-type-specific investigation in biological systems.
View details for DOI 10.1016/j.cell.2022.08.019
View details for PubMedID 36113428
Dendritic calcium signals in rhesus macaque motor cortex drive an optical brain-computer interface.
2021; 12 (1): 3689
Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes newopportunities for studying motor control and designing BCIsvia two photon imaging.
View details for DOI 10.1038/s41467-021-23884-5
View details for PubMedID 34140486
Deep posteromedial cortical rhythm in dissociation.
Advanced imaging methods now allow cell-type-specific recording of neural activity across the mammalian brain, potentially enabling the exploration of how brain-wide dynamical patterns give rise to complex behavioural states1-12. Dissociation is an altered behavioural state in which the integrity of experience is disrupted, resulting in reproducible cognitive phenomena including the dissociation of stimulus detection from stimulus-related affective responses. Dissociation can occur as a result of trauma, epilepsy or dissociative drug use13,14, but despite its substantial basic and clinical importance, the underlying neurophysiology of this state is unknown. Here we establish such a dissociation-like state in mice, induced by precisely-dosed administration of ketamine or phencyclidine. Large-scale imaging of neural activity revealed that these dissociative agents elicited a 1-3-Hz rhythm in layer5 neurons of the retrosplenial cortex. Electrophysiological recording with four simultaneously deployed high-density probes revealed rhythmic coupling of the retrosplenial cortex with anatomically connected components of thalamus circuitry, but uncoupling from most other brain regions was observed-including a notable inverse correlation with frontally projecting thalamic nuclei. In testing for causal significance, we found thatrhythmic optogenetic activation of retrosplenial cortex layer5 neurons recapitulated dissociation-like behavioural effects. Local retrosplenial hyperpolarization-activated cyclic-nucleotide-gated potassium channel 1 (HCN1) pacemakers were required for systemic ketamine to induce this rhythm and to elicit dissociation-like behavioural effects. In a patient with focal epilepsy, simultaneous intracranial stereoencephalography recordings from across the brain revealed a similarly localized rhythm in the homologous deep posteromedial cortex that was temporally correlated with pre-seizure self-reported dissociation, and local brief electrical stimulation of this region elicited dissociative experiences. These results identify themolecular, cellular and physiological properties of a conserved deep posteromedial cortical rhythm that underlies states of dissociation.
View details for DOI 10.1038/s41586-020-2731-9
View details for PubMedID 32939091
Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals.
The resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g., multiple gene expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically targeted technology for writing in and reading out activity patterns for cells in living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically encoded Ca2+ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, we develop a comprehensive intersectional targeting toolbox including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply defined cell types.
View details for DOI 10.1016/j.neuron.2020.06.003
View details for PubMedID 32574559
Three-dimensional intact-tissue sequencing of single-cell transcriptional states
2018; 361 (6400): 380-+
View details for DOI 10.1126/science.aat5691;eaat5691
View details for Web of Science ID 000439923700038
An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.
We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.
View details for PubMedID 28846090
Ancestral Circuits for the Coordinated Modulation of Brain State.
2017; 171 (6): 1411–23.e17
Internal states of the brain profoundly influence behavior. Fluctuating states such as alertness can be governed by neuromodulation, but the underlying mechanisms and cell types involved are not fully understood. We developed a method to globally screen for cell types involved in behavior by integrating brain-wide activity imaging with high-content molecular phenotyping and volume registration at cellular resolution. We used this method (MultiMAP) to record from 22 neuromodulatory cell types in behaving zebrafish during a reaction-time task that reports alertness. We identified multiple monoaminergic, cholinergic, and peptidergic cell types linked to alertness and found that activity in these cell types was mutually correlated during heightened alertness. We next recorded from and controlled homologous neuromodulatory cells in mice; alertness-related cell-type dynamics exhibited striking evolutionary conservation and modulated behavior similarly. These experiments establish a method for unbiased discovery of cellular elements underlying behavior and reveal an evolutionarily conserved set of diverse neuromodulatory systems that collectively govern internal state.
View details for PubMedID 29103613
In Vivo Interrogation of Spinal Mechanosensory Circuits.
2016; 17 (6): 1699-1710
Spinal dorsal horn circuits receive, process, and transmit somatosensory information. To understand how specific components of these circuits contribute to behavior, it is critical to be able to directly modulate their activity in unanesthetized in vivo conditions. Here, we develop experimental tools that enable optogenetic control of spinal circuitry in freely moving mice using commonly available materials. We use these tools to examine mechanosensory processing in the spinal cord and observe that optogenetic activation of somatostatin-positive interneurons facilitates both mechanosensory and itch-related behavior, while reversible chemogenetic inhibition of these neurons suppresses mechanosensation. These results extend recent findings regarding the processing of mechanosensory information in the spinal cord and indicate the potential for activity-induced release of the somatostatin neuropeptide to affect processing of itch. The spinal implant approach we describe here is likely to enable a wide range of studies to elucidate spinal circuits underlying pain, touch, itch, and movement.
View details for DOI 10.1016/j.celrep.2016.10.010
View details for PubMedID 27806306
Optogenetic and chemogenetic strategies for sustained inhibition of pain.
2016; 6: 30570-?
Spatially targeted, genetically-specific strategies for sustained inhibition of nociceptors may help transform pain science and clinical management. Previous optogenetic strategies to inhibit pain have required constant illumination, and chemogenetic approaches in the periphery have not been shown to inhibit pain. Here, we show that the step-function inhibitory channelrhodopsin, SwiChR, can be used to persistently inhibit pain for long periods of time through infrequent transdermally delivered light pulses, reducing required light exposure by >98% and resolving a long-standing limitation in optogenetic inhibition. We demonstrate that the viral expression of the hM4D receptor in small-diameter primary afferent nociceptor enables chemogenetic inhibition of mechanical and thermal nociception thresholds. Finally, we develop optoPAIN, an optogenetic platform to non-invasively assess changes in pain sensitivity, and use this technique to examine pharmacological and chemogenetic inhibition of pain.
View details for DOI 10.1038/srep30570
View details for PubMedID 27484850
View details for PubMedCentralID PMC4971509
The use of optical clearing and multiphoton microscopy for investigation of three-dimensional tissue-engineered constructs.
Tissue engineering. Part C, Methods
2014; 20 (7): 570-7
Recent advances in three-dimensional (3D) tissue engineering have concomitantly generated a need for new methods to visualize and assess the tissue. In particular, methods for imaging intact volumes of whole tissue, rather than a single plane, are required. Herein, we describe the use of multiphoton microscopy, combined with optical clearing, to noninvasively probe decellularized lung extracellular matrix scaffolds and decellularized, tissue-engineered blood vessels. We also evaluate recellularized lung tissue scaffolds. In addition to nondestructive imaging of tissue volumes greater than 4 mm(3), the lung tissue can be visualized using three distinct signals, combined or singly, that allow for simple separation of cells and different components of the extracellular matrix. Because the 3D volumes are not reconstructions, they do not require registration algorithms to generate digital volumes, and maintenance of isotropic resolution is not required when acquiring stacks of images. Once a virtual volume of tissue is generated, structures that have innate 3D features, such as the lumens of vessels and airways, are easily animated and explored in all dimensions. In blood vessels, individual collagen fibers can be visualized at the micron scale and their alignment assessed at various depths through the tissue, potentially providing some nondestructive measure of vessel integrity and mechanics. Finally, both the lungs and vessels assayed here were optically cleared, imaged, and visualized in a matter of hours, such that the added benefits of these techniques can be achieved with little more hassle or processing time than that associated with traditional histological methods.
View details for DOI 10.1089/ten.TEC.2013.0538
View details for PubMedID 24251630
View details for PubMedCentralID PMC4074743
Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs
JOURNAL OF BIOMEDICAL OPTICS
2011; 16 (10)
Multiphoton microscopy of cleared tissue has previously been demonstrated to generate large three-dimensional (3D) volumetric image data on entire intact mouse organs using intrinsic tissue fluorescence. This technique holds great promise for performing 3D virtual biopsies, providing unique information on tissue morphology, and guidance for subsequent traditional slicing and staining. Here, we demonstrate the use of fluorescence lifetime imaging in cleared organs for achieving molecular contrast that can reveal morphologically distinct structures, even in the absence of knowledge of the underlying molecular source. In addition, we demonstrate the power of multimodal imaging, combining multiphoton fluorescence, second harmonic generation, and lifetime imaging to reveal exceptional morphological detail in an optically cleared mouse knee.
View details for DOI 10.1117/1.3641992
View details for Web of Science ID 000297465200016
View details for PubMedID 22029356