Person: Hochbaum, Daniel
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Hochbaum
First Name
Daniel
Name
Hochbaum, Daniel
5 results
Search Results
Now showing 1 - 5 of 5
Publication Single-Cell Analysis of Experience-Dependent Transcriptomic States in Mouse Visual Cortex(2017) Hrvatin, Sinisa; Hochbaum, Daniel; Nagy, M. Aurel; Cicconet, Marcelo; Robertson, Keiramarie; Cheadle, Lucas; Zilionis, Rapolas; Ratner, Alex; Borges-Monroy, Rebeca; Klein, Allon; Sabatini, Bernardo; Greenberg, MichaelActivity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders.Publication Bright and Fast Multicoloured Voltage Reporters via Electrochromic FRET(Nature Publishing Group, 2014) Zou, Peng; Zhao, Yongxin; Douglass, Adam D.; Hochbaum, Daniel; Brinks, Daan; Werley, Christopher A.; Harrison, D. Jed; Campbell, Robert E.; Cohen, AdamGenetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means. We present a palette of multicoloured brightly fluorescent genetically encoded voltage indicators with sensitivities from 8–13% ΔF/F per 100 mV, and half-maximal response times from 4–7 ms. A fluorescent protein is fused to an archaerhodopsin-derived voltage sensor. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. Through a library screen, we identify linkers and fluorescent protein combinations that report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. The freedom to choose a voltage indicator from an array of colours facilitates multicolour voltage imaging, as well as combination with other optical reporters and optogenetic actuators.Publication Flash Memory: Photochemical Imprinting of Neuronal Action Potentials onto a Microbial Rhodopsin(American Chemical Society, 2014) Venkatachalam, Veena; Brinks, Daan; Maclaurin, Dougal; Hochbaum, Daniel; Kralj, Joel; Cohen, AdamWe developed a technique, “flash memory”, to record a photochemical imprint of the activity state—firing or not firing—of a neuron at a user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two nonfluorescent states, D1 and D2, exist in a voltage-dependent equilibrium. A stable fluorescent state, F, is reached by a photochemical conversion from D2. When exposed to light of a wavelength λwrite, population transfers from D2 to F, at a rate determined by the D1 ⇌ D2 equilibrium. The population of F maintains a record of membrane voltage which persists in the dark. Illumination at a later time at a wavelength λread excites fluorescence of F, probing this record. An optional third flash at a wavelength λreset converts F back to D2, for a subsequent write–read cycle. The flash memory method offers the promise to decouple the recording of neural activity from its readout. In principle, the technique may enable one to generate snapshots of neural activity in a large volume of neural tissue, e.g., a complete mouse brain, by circumventing the challenge of imaging a large volume with simultaneous high spatial and high temporal resolution. The proof-of-principle flash memory sensors presented here will need improvements in sensitivity, speed, brightness, and membrane trafficking before this goal can be realized.Publication Bringing bioelectricity to light: all-optical electrophysiology using microbial rhodopsins(2014-10-21) Hochbaum, Daniel; Cohen, Adam Ezra; Cohen, Adam; Hu, Evelyn; Needleman, DanielMy work has focused on the development and application of fluorescent voltage-sensitive proteins based on microbial rhodopsins. These probes led to the discovery of electrical activity in the bacterium Escherichia coli, the first robust optical recordings of action potentials (APs) in mammalian neurons using a genetically encoded voltage reporter, and the development of a genetically targetable all-optical electrophysiology system. I first introduce an engineered fluorescent voltage sensor based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated. I then present another microbial rhodopsin, Archaerhodopsin 3 (Arch), whose endogenous fluorescence exhibited a twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. In rat hippocampal neurons, Arch detected single electrically triggered APs with an optical signal-to-noise ratio > 10. A mutant, Arch(D95N), lacked endogenous proton pumping and had 50% greater sensitivity than the wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual APs. Finally, I introduce evolved archaerhodopsin-based voltage indicators and a spectrally orthogonal channelrhodopsin actuator, which together enabled all-optical electrophysiology. A directed evolution screen yielded two mutants, QuasAr1 and QuasAr2, that showed improved brightness and voltage sensitivity relative to previous archaerhodopsin-based sensors, and microsecond response times. An engineered channelrhodopsin actuator, CheRiff, showed high light sensitivity and rapid kinetics. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured neurons, the Optopatch system probed membrane voltage across temporal and spatial scales, from the sub-cellular and sub-millisecond dynamics of AP propagation, to the simultaneous measurement of firing patterns of many neurons in a circuit. In brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. In human stem cell-derived neurons, Optopatch measurements revealed homeostatic tuning of intrinsic excitability, a subtle form of plasticity that had yet to be observed in human neurons. The suite of tools and techniques presented here enable high-throughput, genetically targeted, and spatially resolved electrophysiology without the use of conventional electrodes.Publication All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins(2014) Hochbaum, Daniel; Zhao, Yongxin; Farhi, Samouil; Klapoetke, Nathan; Werley, Christopher A.; Kapoor, Vikrant; Zou, Peng; Kralj, Joel M.; Maclaurin, Dougal; Smedemark-Margulies, Niklas; Saulnier, Jessica; Boulting, Gabriella; Straub, Christoph; Cho, Yong Ku; Melkonian, Michael; Wong, Gane Ka-Shu; Harrison, D. Jed; Murthy, Venkatesh; Sabatini, Bernardo; Boyden, Edward S.; Campbell, Robert E.; Cohen, AdamAll-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and 2, which show improved brightness and voltage sensitivity, microsecond response times, and produce no photocurrent. We engineered a novel channelrhodopsin actuator, CheRiff, which shows improved light sensitivity and kinetics, and spectral orthogonality to the QuasArs. A co-expression vector, Optopatch, enabled crosstalk-free genetically targeted all-optical electrophysiology. In cultured neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials in dendritic spines, synaptic transmission, sub-cellular microsecond-timescale details of action potential propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In brain slice, Optopatch induced and reported action potentials and subthreshold events, with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without use of conventional electrodes.