Optogenetic strategies for high-efficiency all-optical interrogation using blue light-sensitive opsins
Abstract
All-optical methods for imaging and manipulating brain networks with high spatial resolution are fundamental to study how neuronal ensembles drive behavior. Stimulation of neuronal ensembles using holographic techniques requires high-sensitivity actuators to avoid photodamage and heating. Moreover, two-photon-excitable opsins should be insensitive to light at wavelengths used for imaging. To achieve this goal, we developed a novel soma-targeted variant of the large-conductance blue light-sensitive opsin CoChR (stCoChR). In the mouse cortex in vivo, we combined holographic two-photon stimulation of stCoChR with an amplified laser tuned at the opsin absorption peak and imaging of the red-shifted indicator jRCaMP1a. Compared to previously characterized blue light-sensitive soma-targeted opsins in vivo, stCoChR allowed neuronal stimulation with more than 10-fold lower average power and no spectral crosstalk. The combination of stCoChR, tuned amplified laser stimulation, and red-shifted functional indicators promises to be a powerful tool for large-scale interrogation of neural networks in the intact brain.
Data availability
We provide Source Data for the data plotted in all figures as Excel Source Data files.
Article and author information
Author details
Funding
H2020 European Research Council (647725)
- Tommaso Fellin
Candice Appleton Family Trust
- Ofer Yizhar
H2020-RIA (DEEPER 101016787)
- Ofer Yizhar
NIH Brain Initiative (U01 NS090576)
- Tommaso Fellin
NIH Brain Initiative (U19 NS107464)
- Tommaso Fellin
H2020-RIA (DEEPER 101016787)
- Tommaso Fellin
H2020 European Research Council (819496)
- Ofer Yizhar
Human Frontier Science Program
- Ofer Yizhar
Brain and Behavior Research Foundation
- Ofer Yizhar
Ilse Katz Institute for Material Sciences and Magnetic Resonance Research
- Ofer Yizhar
Adelis Prize for Brain Research
- Ofer Yizhar
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All experiments involving animals were approved by the IIT Animal Welfare Body, by theNational Council on Animal Care of the Italian Ministry of Health (authorization #34/2015-PR, #1084/2020-PR), and by the Institutional Animal Care and Use Committee at the Weizmann Institute of Science, and carried out in accordance with the guidelines established by the European Communities Council Directive.
Copyright
© 2021, Forli et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,336
- views
-
- 601
- downloads
-
- 29
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Developmental Biology
- Neuroscience
Environmental insults, including mild head trauma, significantly increase the risk of neurodegeneration. However, it remains challenging to establish a causative connection between early-life exposure to mild head trauma and late-life emergence of neurodegenerative deficits, nor do we know how sex and age compound the outcome. Using a Drosophila model, we demonstrate that exposure to mild head trauma causes neurodegenerative conditions that emerge late in life and disproportionately affect females. Increasing age-at-injury further exacerbates this effect in a sexually dimorphic manner. We further identify sex peptide signaling as a key factor in female susceptibility to post-injury brain deficits. RNA sequencing highlights a reduction in innate immune defense transcripts specifically in mated females during late life. Our findings establish a causal relationship between early head trauma and late-life neurodegeneration, emphasizing sex differences in injury response and the impact of age-at-injury. Finally, our findings reveal that reproductive signaling adversely impacts female response to mild head insults and elevates vulnerability to late-life neurodegeneration.
-
- Neuroscience
Neurotensin (Nts) is a neuropeptide acting as a neuromodulator in the brain. Pharmacological studies have identified Nts as a potent hypothermic agent. The medial preoptic area, a region that plays an important role in the control of thermoregulation, contains a high density of neurotensinergic neurons and Nts receptors. The conditions in which neurotensinergic neurons play a role in thermoregulation are not known. In this study, optogenetic stimulation of preoptic Nts neurons induced a small hyperthermia. In vitro, optogenetic stimulation of preoptic Nts neurons resulted in synaptic release of GABA and net inhibition of the preoptic pituitary adenylate cyclase-activating polypeptide (Adcyap1) neurons firing activity. GABA-A receptor antagonist or genetic deletion of Slc32a1 (VGAT) in Nts neurons unmasked also an excitatory effect that was blocked by a Nts receptor 1 antagonist. Stimulation of preoptic Nts neurons lacking Slc32a1 resulted in excitation of Adcyap1 neurons and hypothermia. Mice lacking Slc32a1 expression in Nts neurons presented changes in the fever response and in the responses to heat or cold exposure as well as an altered circadian rhythm of body temperature. Chemogenetic activation of all Nts neurons in the brain induced a 4–5°C hypothermia, which could be blocked by Nts receptor antagonists in the preoptic area. Chemogenetic activation of preoptic neurotensinergic projections resulted in robust excitation of preoptic Adcyap1 neurons. Taken together, our data demonstrate that endogenously released Nts can induce potent hypothermia and that excitation of preoptic Adcyap1 neurons is the cellular mechanism that triggers this response.