Dr. Gradinaru's work has focused on developing and using optogenetics (Gradinaru et al., Cell, 2010) and CLARITY (Chung et al., Nature, 2013; Yang et al., Cell, 2014; Treweek et al., Nature Protocols, 2015) to dissect the circuitry underlying neurological disorders such as Parkinson's (Gradinaru et al., Science, 2009). Her group is now working to understand how perturbations of neuronal network activity can permanently impact the function and even viability of comprising neurons and ultimately change network properties and animal behavior. Of particular interest to the group are chronic experiences, subtle but persistent actions on brain networks that can cause lasting changes in the function of individual cells and circuits. Examples include depressive states (it takes weeks of exposure to modest but repeating nuisances to generate an animal model of depression) or Deep Brain Stimulation as used in brain disorders, where electrical stimulation of defined brain areas can improve behavior and this effect can, remarkably, outlive the stimulation. The mechanisms by which these activity changes have long-lasting effects could involve any or all of: (1) circuit rewiring via strengthening and/or weakening of synapses; (2) inducing or preventing neuronal degradation; (3) releasing or blocking protective factors known to aid in neuronal function and health. Research on these topics has been complicated by the heterogeneous nature of the brain. We have previously helped develop optical inhibitors and potentiators of neuronal activity and the ability to target them to defined pathways in the brain as well as the methods necessary to monitor the influence of such manipulations. Our lab will continue to develop enabling technologies for anatomical mapping and chronic bidirectional control to define circuit changes that affect cell function and health and understand the fundamental mechanisms behind such changes.
(1) Methods for precise neuromodulation (Optogenetics): Optogenetics uses light together with genetically encoded, light-sensitive proteins to modulate or monitor the function of specific cell types within living heterogeneous tissue. Optogenetics provided neuroscientists with means to activate or inhibit the firing of a defined population of neurons at physiological, millisecond time scales and study the effects on living, freely moving organisms. By targeting expression of the opsin(s) to a defined class of neuron within a specific brain structure, it is possible to understand the function of those neurons with a precision not possible with electrical or chemical methods. Through optogenetic experiments, neuroscientists are rapidly unraveling how individual neurons and circuits work together to control mood, learning, memory, desire and sensory and motor function as well as how these circuits are altered in disease states. Although now a mature field, in the early days starting with 2005 there were significant challenges that we have solved: many opsins, especially pumps, were not well tolerated by mammalian cells and therefore could not be used in vivo. We have worked out subcellular and transcellular trafficking strategies that resulted in potent and safe optogenetic tools, which includes inhibitors that span the visible spectrum, and generalizable strategies for targeting cells based not on genetic identity, but on morphology or tissue topology, to allow versatile targeting when promoters are not known or in genetically intractable organisms. The same principles used to better traffic opsins outside of the endoplasmic reticulum and into the membrane work for an array of newly described opsins for neuroscience and are likely to help with tolerability in mammalian cells of microbial opsins yet to be discovered or engineered.
a. Gradinaru V, Thompson KR, Zhang F, Mogri M, Kay K, Schneider MB, Deisseroth K. Targeting and readout strategies for fast optical neural control in vitro and in vivo. J Neurosci. 2007 Dec 26;27(52):14231-8. PubMed PMID: 18160630. Download.
b. Zhang F, Gradinaru V (co-first), Adamantidis AR, Durand R, Airan RD, de Lecea L, Deisseroth K. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat Protoc. 2010 Mar;5(3):439-56. PubMed PMID: 20203662; NIHMSID: 194433. Download.
c. Gradinaru V, Zhang F, Ramakrishnan C, Mattis J, Prakash R, Diester I, Goshen I, Thompson KR, Deisseroth K. Molecular and cellular approaches for diversifying and extending optogenetics. Cell. 2010 Apr 2;141(1):154-65. PMC4160532. F1000 must read. Download.
d. Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K. Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat Methods. 2011 Dec 18;9(2):159-72. PubMed PMID: 22179551; PubMed Central PMCID: PMC4165888. Download.
e. Bedbrook, C.N.; Kato, M.; Kumar, S.R.; Lakshmanan, A.; Nath, R.D.; Sun, F.; Sternberg, P.W.; Arnold, F.H.; Gradinaru, V., Genetically encoded spy peptide fusion system to detect plasma membrane-localized proteins in vivo. Chem Biol, 2015; doi:10.1016/j.chembiol.2015.06.020. Download.
(2) Mechanisms of Deep Brain Stimulation by optogenetic deconstruction of diseased brain circuitry: Deep brain stimulation (DBS) is a powerful therapeutic option for intractable movement and affective disorders. The benefits of DBS are immediate and dramatic, manifested as instantaneous improvements in motor function in the case of PD patients. However, due to the nonspecificity of electrical stimulation, the mechanisms behind the effects of DBS are still highly controversial. In order to understand the role of specific cell types underlying effective DBS treatment, we have successfully: (1) developed and optimized optogenetic technologies (molecular and hardware) for safe and effective use in behaving mammals, including readout methods such as the optrode; and (2) employed the above developed optogenetic toolkit to deconstructing diseased brain circuitry, with focus on Parkinson's disease. This work challenged the traditional perception that deep brain stimulation acts mainly by inhibiting local cell bodies at the stimulation site by showing that controlling axons in the stimulation area was sufficient to restore motor behavior in parkinsonian animals. The framework and tools developed are generalizable across many brain circuits to study intact and disordered brain processes and were indeed used subsequently by many projects within our group and outside.
a. Gradinaru V, Mogri M, Thompson KR, Henderson JM, Deisseroth K. Optical deconstruction of parkinsonian neural circuitry. Science. 2009 Apr 17;324(5925):354-9. PubMed PMID: 19299587. Science News Focus. F1000 exceptional. Download.
b. Witten IB, Lin SC, Brodsky M, Prakash R, Diester I, Anikeeva P, Gradinaru V, Ramakrishnan C, Deisseroth K. Cholinergic interneurons control local circuit activity and cocaine conditioning. Science. 2010 Dec 17;330(6011):1677-81. PubMed PMID: 21164015; PubMed Central PMCID: PMC3142356. Download.
c. Tye KM, Prakash R, Kim SY, Fenno LE, Grosenick L, Zarabi H, Thompson KR, Gradinaru V, Ramakrishnan C, Deisseroth K. Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature. 2011 Mar 17;471(7338):358-62. PubMed PMID: 21389985; PMC3154022. Download.
d. Domingos AI, Vaynshteyn J, Voss HU, Ren X, Gradinaru V, Zang F, Deisseroth K, de Araujo IE, Friedman J. Leptin regulates the reward value of nutrient. Nat Neurosci. 2011 Nov 13;14(12):1562-8. PubMed PMID: 22081158; PubMed Central PMCID: PMC4238286. Download.
(3) Methods for anatomical mapping of intact circuits (Tissue Clearing, CLARITY): Even with the power of optogenetics for control and readout of brain networks, a standing challenge is knowing which circuits to modulate for an intended therapeutic effect: we do not have detailed maps of connectivity across large brain volumes. This can be a serious problem, as our DBS optogenetic study above showed. That electrical DBS might act fundamentally through white matter away from the electrode site highlights the need for better brain maps. It is however difficult to create such maps for phenotypically distinct fine axons that run in bundles throughout the brain when the traditional method involves sectioning the tissue in paper-thin slices, imaging each slice, and putting it all back together with imaging software: it is slow, tedious, costly, and error prone. We invested in tissue clearing instead to remove the lipids, which obstruct the view, and created a new method known as CLARITY (Nature, 2013), which renders the tissue transparent for easy visualization and identification of cellular components and their molecular identity without slicing. Tissue clearing complements optogenetics, in that it can reveal, with ease, circuit-wide effects of optogenetic manipulations and also aid in mapping novel circuits that need tuning in disease. In an attempt to perfect the execution of CLARITY, our group has recently reported (Cell, 2014) the first case of whole-body clearing – transparent rodents that can be used to obtain detailed maps of both central and peripheral nerves at their target organs throughout the body. Such nerves could then be modulated with optogenetics in animal models of disease to understand what needs tuning to improve symptoms and the resulting knowledge could facilitate better therapies that rely on, for example, electrically stimulating nerves for better organ function or for decreasing chronic pain.
a. Deisseroth, K., Gradinaru, V. "Functional Targeted Brain Endoskeletonization," US Patent App. 13/980,842, 2012. Download.
b. Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K. Structural and molecular interrogation of intact biological systems. Nature. 2013 May 16;497(7449):332-7. PubMed PMID: 23575631; PMC4092167. Download.
c. Birmingham K, Gradinaru V, Anikeeva P, Grill WM, Pikov V, McLaughlin B, Pasricha P, Weber D, Ludwig K, Famm K. Bioelectronic medicines: a research roadmap. Nat Rev Drug Discov. 2014 Jun;13(6):399-400. PubMed PMID: 24875080. Download.
d. Yang B, Treweek JB, Kulkarni RP, Deverman BE, Chen CK, Lubeck E, Shah S, Cai L, Gradinaru V. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell. 2014 Aug 14;158(4):945-58. PMC4153367. Highlighted by NIH, Nature, Science, F1000. Scientific American 10 World Changing Ideas 2014. Nature Biotechnology News and Views. Download.
e. Treweek, J.B.; Chan, K.Y.; Flytzanis, N.C.; Yang, B.; Deverman, B.E.; Greenbaum, A.; Lignell, A.; Xiao, C.; Cai, L.; Ladinsky, M.S.; Bjorkman, P.J.; Fowlkes, C.C.; Gradinaru, V. Whole-Body Tissue Stabilization and Selective Extractions via Tissue-Hydrogel Hybrids for High Resolution Intact Circuit Mapping and Phenotyping. Nature Protocols (2015). Download.
(4) Methods for optical monitoring of neuronal activity: In addition to optogenetic control of neuronal activity we need feedback on how exactly the tissue is responding to light modulation in real-time, and tune it up or down accordingly – for example to stop seizures. We have worked on two related topics: Optogenetic fMRI and Optical Voltage Sensors. While at Stanford in the group of Prof. Karl Deisseroth Dr. Gradinaru helped facilitate the combination of Optogenetics and fMRI for the first time to obtain unprecedented ability to both control and monitor neuronal activity over large assemblies of neurons in vivo. The research showed that controlling the cortico-thalamic excitatory neurons specifically, either at the cell body or at the axons, resulted in robust fMRI signal therefore conclusively linking changes in defined neuronal populations to the BOLD fMRI signal. This work opened up exciting avenues for all optical control and monitoring of the brain in intact living animals. The Gradinaru group at Caltech now works on voltage sensing with microbial opsins for all-optical control and readout of neuronal networks. Opsins can be engineered for diverse properties, including increased opsin radiance whose level tracks the membrane voltage changes with high temporal precision in defined cell types. Our group in collaboration with Dr. Frances Arnold recently used directed evolution of opsins to make them better at reporting action potentials. We hope to further combine optogenetic sensors and actuators to modulate and read activity in DBS paradigms.
a. Lee JH, Durand R, Gradinaru V, Zhang F, Goshen I, Kim DS, Fenno LE, Ramakrishnan C, Deisseroth K. Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Nature. 2010 Jun 10;465(7299):788-92. PubMed PMID: 20473285; PubMed Central PMCID: PMC3177305. Nature News and Views. F1000 exceptional. Download.
b. McIsaac RS, Engqvist MK, Wannier T, Rosenthal AZ, Herwig L, Flytzanis NC, Imasheva ES, Lanyi JK, Balashov SP, Gradinaru V, Arnold FH. Directed evolution of a far-red fluorescent rhodopsin. Proc Natl Acad Sci U S A. 2014 Sep 9;111(36):13034-9. PMC4246972. Download.
c. Flytzanis NC, Bedbrook CN, Chiu H, Engqvist MK, Xiao C, Chan KY, Sternberg PW, Arnold FH, Gradinaru V. Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons. Nat Commun. 2014 Sep 15;5:4894. PMC4166526. Selected by Nature Methods for "Methods to Watch." Cited 12 times. Download.
d. Bedbrook, C.N.; Kato, M.; Kumar, S.R.; Lakshmanan, A.; Nath, R.D.; Sun, F.; Sternberg, P.W.; Arnold, F.H.; Gradinaru, V., Genetically encoded spy peptide fusion system to detect plasma membrane-localized proteins in vivo. Chem Biol, 2015; doi:10.1016/j.chembiol.2015.06.020. Download
(5) Viral-based approaches to non-invasive whole-brain cargo delivery: Genetically-encoded tools that can be used to visualize, monitor, and modulate mammalian neurons are revolutionizing neuroscience. These tools are particularly powerful in rodents and invertebrate models where intersectional transgenic strategies are available to restrict their expression to defined cell populations. However, use of genetic tools in non-transgenic animals is often hindered by the lack of vectors capable of safe, efficient, and specific delivery to the desired cellular targets. To begin to address these challenges, we have developed an in vivo Cre-based selection platform (CREATE) for identifying adeno-associated viruses (AAVs) that more efficiently transduce genetically defined cell populations. Our platform's novelty and power arises from the additional selective pressure imparted by a recovery step that amplifies only those capsid variants that have functionally transduced a genetically-defined, Cre-expressing target cell population. The Cre-dependent capsid recovery works within heterogeneous tissue samples without the need for additional steps such as selective capsid recovery approaches that require cell sorting or secondary adenovirus infection. As a first test of the CREATE platform, we selected for viruses that transduced the brain after intravascular delivery and found a novel vector, AAV-PHP.B, that is 40- to 90-fold more efficient at transducing the brain than the current standard, AAV9. AAV-PHP.B transduces most neuronal types and glia across the brain. We also demonstrate here how whole-body tissue clearing can facilitate transduction maps of systemically delivered genes. Since CNS disorders are notoriously challenging due to the restrictive nature of the blood brain barrier our discovery that recombinant vectors can be engineered to overcome this barrier is enabling for the whole field. With the exciting advances in gene editing via the CRISPR-Cas, RNA interference and gene replacement strategies, the availability of potent gene delivery methods provided by vectors such as our reported AAV-PHP.B is key to advancing the field of genome engineering.
a. Deverman BE, Pravdo P, Simpson B, Banerjee A, Kumar, S.R., Chan K, Wu WL, Yang B, Gradinaru V. Cre-Dependent Capsid Selection Yields AAVs for Global Gene Transfer to the Adult Brain. Nature Biotechnol (2015). Download.
Dr. Gradinaru was recently named a Heritage Principal Investigator.